Modulators of PCSK9 Expression

ABSTRACT

The present embodiments provide methods, compounds, and compositions useful for inhibiting PCSK9 expression, which may be useful for treating, preventing, or ameliorating a disease associated with PCSK9.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled200615 SequenceListing.xml, created Feb. 2, 2023, which is 2,018 kb insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositionsuseful for inhibiting proprotein convertase subtilisin/kexin type 9(PCSK9) expression, and in certain instances, reducing the amount ofPCSK9 protein in a cell or animal, which can be useful for treating,preventing, or ameliorating a disease associated with PCSK9.

BACKGROUND

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme withan important role in lipoprotein metabolism. Rare gain-of-functionmutations in PCSK9 lead to a high LDL-C level and premature coronaryheart disease, whereas loss-of-function variants lead to a low LDL-Clevel and a reduced incidence of coronary heart disease (Zhao et al.,Am. J. Hum. Genet. 2006, 79: 514-523; Horton et al., J. Lipid Res. 2009,50: Suppl: S172-S177). Therefore, PCSK9 is a well-validated target forLDL-choleserol-lowering therapy (Hooper et al., Expert Opin. Biol. Ther.2013, 13: 429-435).

Antibodies for blocking PCSK9, Alirocumab and Evolocumab, have beendemonstrated to reduce circulating PCSK9 levels and lowerLDL-cholesterol levels but have a short duration of action,necessitating frequent subcutaneous injections (Zhang et al., BMC Med.2015, 13: 123; Navarese et al., Ann. Intern. Med. 2015, 163: 40-51).

It is an object herein to provide compounds, methods, and pharmaceuticalcompositions for the improved treatment of diseases such ascardiovascular diseases, dyslipidemias, mixed dyslipidemias, andhypercholesterolemia.

SUMMARY

Provided herein are compounds and methods for reducing the amount oractivity of PCSK9 mRNA, and in certain embodiments, reducing the amountof PCSK9 protein in a cell or animal. In certain embodiments, the animalhas a cardiovascular disease. In certain embodiments, the disease isdyslipidemia. In certain embodiments, the disease is mixed dyslipidemia.In certain embodiments, the disease is hypercholesterolemia. Certaincompounds provided herein are directed to compounds and compositionsthat reduce LDL-cholesterol in an animal.

Certain embodiments provided herein are directed to potent and tolerablecompounds and compositions useful for inhibiting PCSK9 expression, whichcan be useful for treating, preventing, ameliorating, or slowingprogression of a cardiovascular disease. Certain embodiments providedherein are directed to compounds and compositions that are more potentor have greater therapeutic value than compounds publicly disclosed.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the embodiments, as claimed. Herein, the useof the singular includes the plural unless specifically statedotherwise. As used herein, the use of “or” means “and/or” unless statedotherwise. Furthermore, the use of the term “including” as well as otherforms, such as “includes” and “included”, is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and GenBank and NCBI reference sequence records arehereby expressly incorporated by reference for the portions of thedocument discussed herein, as well as in their entirety.

It is understood that the sequence set forth in each SEQ ID NO in theexamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, compoundsdefined by a SEQ ID NO may comprise, independently, one or moremodifications to a sugar moiety, an internucleoside linkage, or anucleobase. Compounds described by ISIS number indicate a combination ofnucleobase sequence, chemical modification, and motif.

Definitions

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-deoxynucleoside” means a nucleoside comprising 2′-H(H) furanosylsugar moiety, as found in naturally occurring deoxyribonucleic acids(DNA). In certain embodiments, a 2′-deoxynucleoside may comprise amodified nucleobase or may comprise an RNA nucleobase (uracil).

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification at the 2′ position of a furanosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means anucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” or “2-modified nucleoside” means anucleoside comprising a 2′-substituted or 2′-modified sugar moiety. Asused herein, “2′-substituted” or “2-modified” in reference to a sugarmoiety means a sugar moiety comprising at least one 2′-substituent groupother than H or OH.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular compound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular compound.

“5-methylcytosine” means a cytosine with a methyl group attached to the5 position.

“About” means within +10% of a value. For example, if it is stated, “thecompounds affected about 70% inhibition of PCSK9”, it is implied thatPCSK9 levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing acompound or composition provided herein to an individual to perform itsintended function. An example of a route of administration that can beused includes, but is not limited to parenteral administration, such assubcutaneous, intravenous, or intramuscular injection or infusion.

“Amelioration” refers to an improvement or lessening of at least oneindicator, sign, or symptom of an associated disease, disorder, orcondition. In certain embodiments, amelioration includes a delay orslowing in the progression or severity of one or more indicators of acondition or disease. The progression or severity of indicators may bedetermined by subjective or objective measures, which are known to thoseskilled in the art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable and/or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid compared to target nucleic acid levels ortarget protein levels in the absence of the antisense compound to thetarget.

“Antisense compound” means a compound comprising an oligonucleotide andoptionally one or more additional features, such as a conjugate group orterminal group. Examples of antisense compounds include single-strandedand double-stranded compounds, such as, oligonucleotides, ribozymes,siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels inthe presence of an antisense compound complementary to a target nucleicacid compared to target nucleic acid levels in the absence of theantisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with target nucleic acid, wherein the outcome or effect ofthe hybridization is either target degradation or target occupancy withconcomitant stalling of the cellular machinery involving, for example,transcription or splicing.

“Antisense oligonucleotide” means an oligonucleotide having a nucleobasesequence that is complementary to a target nucleic acid or region orsegment thereof. In certain embodiments, an antisense oligonucleotide isspecifically hybridizable to a target nucleic acid or region or segmentthereof.

“Bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclicsugar moiety. “Bicyclic sugar” or “bicyclic sugar moiety” means amodified sugar moiety comprising two rings, wherein the second ring isformed via a bridge connecting two of the atoms in the first ringthereby forming a bicyclic structure. In certain embodiments, the firstring of the bicyclic sugar moiety is a furanosyl moiety. In certainembodiments, the bicyclic sugar moiety does not comprise a furanosylmoiety.

“Branching group” means a group of atoms having at least 3 positionsthat are capable of forming covalent linkages to at least 3 groups. Incertain embodiments, a branching group provides a plurality of reactivesites for connecting tethered ligands to an oligonucleotide via aconjugate linker and/or a cleavable moiety.

“Cell-targeting moiety” means a conjugate group or portion of aconjugate group that is capable of binding to a particular cell type orparticular cell types.

“cEt” or “constrained ethyl” means a bicyclic furanosyl sugar moietycomprising a bridge connecting the 4′-carbon and the 2′-carbon, whereinthe bridge has the formula: 4′-CH(CH₃)—O-2′.

“cEt nucleoside” means a nucleoside comprising a cEt modified sugarmoiety.

“Chemical modification” in a compound describes the substitutions orchanges through chemical reaction, of any of the units in the compoundrelative to the original state of such unit. “Modified nucleoside” meansa nucleoside having, independently, a modified sugar moiety and/ormodified nucleobase. “Modified oligonucleotide” means an oligonucleotidecomprising at least one modified internucleoside linkage, a modifiedsugar, and/or a modified nucleobase.

“Chemically distinct region” refers to a region of a compound that is insome way chemically different than another region of the same compound.For example, a region having 2′-O-methoxyethyl nucleotides is chemicallydistinct from a region having nucleotides without 2′-O-methoxyethylmodifications.

“Chimeric antisense compounds” means antisense compounds that have atleast 2 chemically distinct regions, each position having a plurality ofsubunits.

“Cleavable bond” means any chemical bond capable of being split. Incertain embodiments, a cleavable bond is selected from among: an amide,a polyamide, an ester, an ether, one or both esters of a phosphodiester,a phosphate ester, a carbamate, a di-sulfide, or a peptide.

“Cleavable moiety” means a bond or group of atoms that is cleaved underphysiological conditions, for example, inside a cell, an animal, or ahuman.

“Complementary” in reference to an oligonucleotide means the nucleobasesequence of such oligonucleotide or one or more regions thereof matchesthe nucleobase sequence of another oligonucleotide or nucleic acid orone or more regions thereof when the two nucleobase sequences arealigned in opposing directions. Nucleobase matches or complementarynucleobases, as described herein, are limited to the following pairs:adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C)and guanine (G), and 5-methyl cytosine (^(m)C) and guanine (G) unlessotherwise specified. Complementary oligonucleotides and/or nucleic acidsneed not have nucleobase complementarity at each nucleoside and mayinclude one or more nucleobase mismatches. By contrast, “fullycomplementary” or “100% complementary” in reference to oligonucleotidesmeans that such oligonucleotides have nucleobase matches at eachnucleoside without any nucleobase mismatches.

“Conjugate group” means a group of atoms that is attached to anoligonucleotide. Conjugate groups include a conjugate moiety and aconjugate linker that attaches the conjugate moiety to theoligonucleotide.

“Conjugate linker” means a group of atoms comprising at least one bondthat connects a conjugate moiety to an oligonucleotide.

“Conjugate moiety” means a group of atoms that is attached to anoligonucleotide via a conjugate linker.

“Contiguous” in the context of an oligonucleotide refers to nucleosides,nucleobases, sugar moieties, or internucleoside linkages that areimmediately adjacent to each other. For example, “contiguousnucleobases” means nucleobases that are immediately adjacent to eachother in a sequence.

“Designing” or “Designed to” refer to the process of designing acompound that specifically hybridizes with a selected nucleic acidmolecule.

“Differently modified” means chemical modifications or chemicalsubstituents that are different from one another, including absence ofmodifications. Thus, for example, a MOE nucleoside and an unmodified DNAnucleoside are “differently modified,” even though the DNA nucleoside isunmodified. Likewise, DNA and RNA are “differently modified,” eventhough both are naturally-occurring unmodified nucleosides.

Nucleosides that are the same but for comprising different nucleobasesare not differently modified. For example, a nucleoside comprising a2′-OMe modified sugar and an unmodified adenine nucleobase and anucleoside comprising a 2′-OMe modified sugar and an unmodified thyminenucleobase are not differently modified.

“Double-stranded antisense compound” means an antisense compoundcomprising two oligomeric compounds that are complementary to each otherand form a duplex, and wherein one of the two said oligomeric compoundscomprises an oligonucleotide.

“Effective amount” means the amount of compound sufficient to effectuatea desired physiological outcome in an individual in need of thecompound. The effective amount may vary among individuals depending onthe health and physical condition of the individual to be treated, thetaxonomic group of the individuals to be treated, the formulation of thecomposition, assessment of the individual's medical condition, and otherrelevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. Such structures include, but are not limited to, the products oftranscription and translation.

“Gapmer” means an oligonucleotide comprising an internal region having aplurality of nucleosides that support RNase H cleavage positionedbetween external regions having one or more nucleosides, wherein thenucleosides comprising the internal region are chemically distinct fromthe nucleoside or nucleosides comprising the external regions. Theinternal region may be referred to as the “gap” and the external regionsmay be referred to as the “wings.” “Hybridization” means the annealingof oligonucleotides and/or nucleic acids. While not limited to aparticular mechanism, the most common mechanism of hybridizationinvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleobases.

In certain embodiments, complementary nucleic acid molecules include,but are not limited to, an antisense compound and a nucleic acid target.In certain embodiments, complementary nucleic acid molecules include,but are not limited to, an oligonucleotide and a nucleic acid target.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements of the same kind (e.g. no interveningnucleobases between the immediately adjacent nucleobases).

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Inhibiting the expression or activity” refers to a reduction orblockade of the expression or activity relative to the expression ofactivity in an untreated or control sample and does not necessarilyindicate a total elimination of expression or activity.

“Internucleoside linkage” means a group or bond that forms a covalentlinkage between adjacent nucleosides in an oligonucleotide. “Modifiedinternucleoside linkage” means any internucleoside linkage other than anaturally occurring, phosphate internucleoside linkage. Non-phosphatelinkages are referred to herein as modified internucleoside linkages.

“Lengthened oligonucleotides” are those that have one or more additionalnucleosides relative to an oligonucleotide disclosed herein, e.g. aparent oligonucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by aninternucleoside linkage.

“Linker-nucleoside” means a nucleoside that links an oligonucleotide toa conjugate moiety. Linker-nucleosides are located within the conjugatelinker of a compound. Linker-nucleosides are not considered part of theoligonucleotide portion of a compound even if they are contiguous withthe oligonucleotide.

“Mismatch” or “non-complementary” means a nucleobase of a firstoligonucleotide that is not complementary to the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligonucleotides are aligned. For example, nucleobasesincluding but not limited to a universal nucleobase, inosine, andhypoxanthine, are capable of hybridizing with at least one nucleobasebut are still mismatched or non-complementary with respect to nucleobaseto which it hybridized. As another example, a nucleobase of a firstoligonucleotide that is not capable of hybridizing to the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligonucleotides are aligned is a mismatch ornon-complementary nucleobase.

“Modulating” refers to changing or adjusting a feature in a cell,tissue, organ or organism. For example, modulating PCSK9 RNA can mean toincrease or decrease the level of PCSK9 RNA and/or PCSK9 protein in acell, tissue, organ or organism. A “modulator” effects the change in thecell, tissue, organ or organism. For example, a PCSK9 compound can be amodulator that decreases the amount of PCSK9 RNA and/or PCSK9 protein ina cell, tissue, organ or organism.

“MOE” means methoxyethyl.

“Monomer” refers to a single unit of an oligomer. Monomers include, butare not limited to, nucleosides and nucleotides.

“Motif” means the pattern of unmodified and/or modified sugar moieties,nucleobases, and/or internucleoside linkages, in an oligonucleotide.

“Natural” or “naturally occurring” means found in nature.

“Non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety”means a modified sugar moiety that comprises a modification, such as asubstituent, that does not form a bridge between two atoms of the sugarto form a second ring.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes, but is not limited to, ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids, anddouble-stranded nucleic acids.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid. As used herein a “naturally occurringnucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), andguanine (G). A “modified nucleobase” is a naturally occurring nucleobasethat is chemically modified. A “universal base” or “universalnucleobase” is a nucleobase other than a naturally occurring nucleobaseand modified nucleobase, and is capable of pairing with any nucleobase.

“Nucleobase sequence” means the order of contiguous nucleobases in anucleic acid or oligonucleotide independent of any sugar orinternucleoside linkage.

“Nucleoside” means a compound comprising a nucleobase and a sugarmoiety. The nucleobase and sugar moiety are each, independently,unmodified or modified. “Modified nucleoside” means a nucleosidecomprising a modified nucleobase and/or a modified sugar moiety.Modified nucleosides include abasic nucleosides, which lack anucleobase.

“Oligomeric compound” means a compound comprising a singleoligonucleotide and optionally one or more additional features, such asa conjugate group or terminal group.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another. Unlessotherwise indicated, oligonucleotides consist of 8-80 linkednucleosides. “Modified oligonucleotide” means an oligonucleotide,wherein at least one sugar, nucleobase, or internucleoside linkage ismodified. “Unmodified oligonucleotide” means an oligonucleotide thatdoes not comprise any sugar, nucleobase, or internucleosidemodification.

“Parent oligonucleotide” means an oligonucleotide whose sequence is usedas the basis of design for more oligonucleotides of similar sequence butwith different lengths, motifs, and/or chemistries. The newly designedoligonucleotides may have the same or overlapping sequence as the parentoligonucleotide.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration.

“Pharmaceutically acceptable carrier or diluent” means any substancesuitable for use in administering to an individual. For example, apharmaceutically acceptable carrier can be a sterile aqueous solution,such as PBS or water-for-injection.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of compounds, such as oligomericcompounds or oligonucleotides, i.e., salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects thereto.

“Pharmaceutical agent” means a compound that provides a therapeuticbenefit when administered to an individual.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more compounds or salt thereof and asterile aqueous solution.

“Phosphorothioate linkage” means a modified phosphate linkage in whichone of the non-bridging oxygen atoms is replaced with a sulfur atom. Aphosphorothioate internucleoside linkage is a modified internucleosidelinkage.

“Phosphorus moiety” means a group of atoms comprising a phosphorus atom.In certain embodiments, a phosphorus moiety comprises a mono-, di-, ortri-phosphate, or phosphorothioate.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an oligomeric compound.

“Prevent” refers to delaying or forestalling the onset, development orprogression of a disease, disorder, or condition for a period of timefrom minutes to indefinitely.

“Prodrug” means a compound in a form outside the body which, whenadministered to an individual, is metabolized to another form within thebody or cells thereof. In certain embodiments, the metabolized form isthe active, or more active, form of the compound (e.g., drug). Typicallyconversion of a prodrug within the body is facilitated by the action ofan enzyme(s) (e.g., endogenous or viral enzyme) or chemical(s) presentin cells or tissues, and/or by physiologic conditions.

“Reduce” means to bring down to a smaller extent, size, amount, ornumber.

“RefSeq No.” is a unique combination of letters and numbers assigned toa sequence to indicate the sequence is for a particular targettranscript (e.g., target gene). Such sequence and information about thetarget gene (collectively, the gene record) can be found in a geneticsequence database. Genetic sequence_databases include the NCBI ReferenceSequence database, GenBank, the European Nucleotide Archive, and the DNAData Bank of Japan (the latter three forming the InternationalNucleotide Sequence Database Collaboration or INSDC).

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“RNAi compound” means an antisense compound that acts, at least in part,through RISC or Ago2, but not through RNase H, to modulate a targetnucleic acid and/or protein encoded by a target nucleic acid. RNAicompounds include, but are not limited to double-stranded siRNA,single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.

“Segments” are defined as smaller or sub-portions of regions within anucleic acid.

“Side effects” means physiological disease and/or conditionsattributable to a treatment other than the desired effects. In certainembodiments, side effects include injection site reactions, liverfunction test abnormalities, renal function abnormalities, livertoxicity, renal toxicity, central nervous system abnormalities,myopathies, and malaise. For example, increased aminotransferase levelsin serum may indicate liver toxicity or liver function abnormality. Forexample, increased bilirubin may indicate liver toxicity or liverfunction abnormality.

“Single-stranded” in reference to a compound means the compound has onlyone oligonucleotide. “Self-complementary” means an oligonucleotide thatat least partially hybridizes to itself. A compound consisting of oneoligonucleotide, wherein the oligonucleotide of the compound isself-complementary, is a single-stranded compound. A single-strandedcompound may be capable of binding to a complementary compound to form aduplex.

“Sites” are defined as unique nucleobase positions within a targetnucleic acid.

“Specifically hybridizable” refers to an oligonucleotide having asufficient degree of complementarity between the oligonucleotide and atarget nucleic acid to induce a desired effect, while exhibiting minimalor no effects on non-target nucleic acids. In certain embodiments,specific hybridization occurs under physiological conditions.

“Specifically inhibit” with reference to a target nucleic acid means toreduce or block expression of the target nucleic acid while exhibitingfewer, minimal, or no effects on non-target nucleic acids. Reductiondoes not necessarily indicate a total elimination of the target nucleicacid's expression.

“Standard cell assay” means assay(s) described in the Examples andreasonable variations thereof.

“Standard in vivo experiment” means the procedure(s) described in theExample(s) and reasonable variations thereof.

“Sugar moiety” means an unmodified sugar moiety or a modified sugarmoiety. “Unmodified sugar moiety” or “unmodified sugar” means a 2′-OH(H)furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), ora 2′-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”).Unmodified sugar moieties have one hydrogen at each of the 1′, 3′, and4′ positions, an oxygen at the 3′ position, and two hydrogens at the 5′position. “Modified sugar moiety” or “modified sugar” means a modifiedfuranosyl sugar moiety or a sugar surrogate. “Modified furanosyl sugarmoiety” means a furanosyl sugar comprising a non-hydrogen substituent inplace of at least one hydrogen of an unmodified sugar moiety. In certainembodiments, a modified furanosyl sugar moiety is a 2′-substituted sugarmoiety. Such modified furanosyl sugar moieties include bicyclic sugarsand non-bicyclic sugars.

“Sugar surrogate” means a modified sugar moiety having other than afuranosyl moiety that can link a nucleobase to another group, such as aninternucleoside linkage, conjugate group, or terminal group in anoligonucleotide. Modified nucleosides comprising sugar surrogates can beincorporated into one or more positions within an oligonucleotide andsuch oligonucleotides are capable of hybridizing to complementarycompounds or nucleic acids.

“PCSK9” means any nucleic acid or protein of PCSK9. “PCSK9 nucleic acid”means any nucleic acid encoding PCSK9. For example, in certainembodiments, a PCSK9 nucleic acid includes a DNA sequence encodingPCSK9, an RNA sequence transcribed from DNA encoding PCSK9 (includinggenomic DNA comprising introns and exons) and an mRNA sequence encodingPCSK9. “PCSK9 mRNA” means an mRNA encoding a PCSK9 protein. The targetmay be referred to in either upper or lower case.

“PCSK9 specific inhibitor” refers to any agent capable of specificallyinhibiting PCSK9 RNA and/or PCSK9 protein expression or activity at themolecular level. For example, PCSK9 specific inhibitors include nucleicacids (including antisense compounds), peptides, antibodies, smallmolecules, and other agents capable of inhibiting the expression ofPCSK9 RNA and/or PCSK9 protein.

“Target gene” refers to a gene encoding a target.

“Targeting” means the specific hybridization of a compound to a targetnucleic acid in order to induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby compounds described herein.

“Target region” means a portion of a target nucleic acid to which one ormore compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which a compound is targeted. “5′ target site” refers to the5′-most nucleotide of a target segment. “3′ target site” refers to the3′-most nucleotide of a target segment.

“Terminal group” means a chemical group or group of atoms that iscovalently linked to a terminus of an oligonucleotide.

“Therapeutically effective amount” means an amount of a compound,pharmaceutical agent, or composition that provides a therapeutic benefitto an individual.

“Treat” refers to administering a compound or pharmaceutical compositionto an animal in order to effect an alteration or improvement of adisease, disorder, or condition in the animal.

Certain Embodiments

Certain embodiments provide methods, compounds and compositions forinhibiting PCSK9 expression.

Certain embodiments provide compounds targeted to a PCSK9 nucleic acid.In certain embodiments, the PCSK9 nucleic acid has the sequence setforth in RefSeq or GENBANK Accession No. NM_174936.3 and GENBANKAccession No. NC_000001.11 truncated from nucleotides 55036001 to55068000 (incorporated by reference, disclosed herein as SEQ ID NO: 1and SEQ ID NO: 2, respectively). In certain embodiments, the compound isan antisense compound or oligomeric compound. In certain embodiments,the compound is single-stranded. In certain embodiments, the compound isdouble-stranded.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 8 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 8 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 10 to 30 linked nucleosidesin length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 9 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 9 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 10 to 30 linked nucleosidesin length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 10 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 10 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 10 to 30 linked nucleosidesin length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 11 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 11 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 11 to 30 linked nucleosidesin length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 12 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 12 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 12 to 30 linked nucleosidesin length.

In certain embodiments, the compound comprises a modifiedoligonucleotide 30 linked nucleosides in length. In certain embodiments,the compound is an antisense compound or oligomeric compound.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 14 to 80 linked nucleosides in length and having anucleobase sequence comprising the nucleobase sequence of any one of SEQID NOs: 3-1540. In certain embodiments, the compound is an antisensecompound or oligomeric compound. In certain embodiments, the compound issingle-stranded. In certain embodiments, the compound isdouble-stranded. In certain embodiments, the modified oligonucleotide is14 to 30 linked nucleosides in length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide consisting of the nucleobase sequence of any one of SEQID NOs: 3-1540. In certain embodiments, the compound is an antisensecompound or oligomeric compound. In certain embodiments, the compound issingle-stranded. In certain embodiments, the compound isdouble-stranded.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 8 to 80 linked nucleosides in length and complementarywithin nucleobases 6356-6371, 12843-12947, 12905-12948, 17681-17700,19653-19673, 27626-27669, 27895-27949, and 28105-28136 of SEQ ID NO: 2,wherein said modified oligonucleotide is at least 85%, at least 90%, atleast 95%, or 100% complementary to SEQ ID NO: 2. In certainembodiments, the compound is an antisense compound or oligomericcompound. In certain embodiments, the compound is single-stranded. Incertain embodiments, the compound is double-stranded. In certainembodiments, the modified oligonucleotide is 10 to 30 linked nucleosidesin length.

Certain embodiments provide a compound comprising a modifiedoligonucleotide 8 to 80 linked nucleosides in length and having anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion of nucleobases6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673,27626-27669, 27895-27949, and 28105-28136 of SEQ ID NO: 2, wherein saidmodified oligonucleotide is at least 85%, at least 90%, at least 95%, or100% complementary to SEQ ID NO: 2. In certain embodiments, the compoundis an antisense compound or oligomeric compound. In certain embodiments,the compound is single-stranded. In certain embodiments, the compound isdouble-stranded. In certain embodiments, the modified oligonucleotide is10 to 30 linked nucleosides in length.

In certain embodiments, compounds target a region of a PCSK9 nucleicacid. In certain embodiments, such compounds targeted to a region of aPCSK9 nucleic acid have a contiguous nucleobase portion that iscomplementary to an equal length nucleobase portion of the region. Forexample, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 contiguous nucleobase portion complementary to anequal length portion of a region recited herein. In certain embodiments,such compounds target the following nucleotide regions of SEQ ID NO: 2:6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673,27626-27669, 27895-27949, and 28105-28136. In certain embodiments, thesecompounds are antisense compounds, oligomeric compounds, oroligonucleotides.

In certain embodiments, a compound comprises a modified oligonucleotide8 to 80 linked nucleosides in length and having at least an 8, 9, 10,11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary toan equal length portion within nucleotides 6356-6371, 12909-12924,17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931,27917-27932, and 28107-28122 of SEQ ID NO: 2. In certain embodiments,the modified oligonucleotide is 10 to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotide8 to 80 linked nucleosides in length and complementary withinnucleotides 6356-6371, 12909-12924, 17685-17700, 19658-19673,27643-27658, 27906-27921, 27916-27931, 27917-27932, and 28107-28122 ofSEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is 10to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotide8 to 80 linked nucleosides in length and having a nucleobase sequencecomprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguousnucleobase portion any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147,1149, 1016, 955, 1195, and 353. In certain embodiments, the modifiedoligonucleotide is 10 to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotide8 to 80 linked nucleosides in length and having a nucleobase sequencecomprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149,1016, 955, 1195, and 353. In certain embodiments, the modifiedoligonucleotide is 10 to 30 linked nucleosides in length.

In certain embodiments, a compound comprises a modified oligonucleotidehaving a nucleobase sequence consisting of any one of SEQ ID NOs: 1016,1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.

In certain embodiments, compounds targeted to PCSK9 are ISIS 863568,ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS863655, ISIS 863670, and ISIS 863681. Out of over 1540 compounds thatwere screened as described in the Examples section below, ISIS 863568,ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS863655, ISIS 863670, and ISIS 863681 emerged as the top lead compounds.In particular, ISIS 863633 exhibited the best combination of propertiesin terms of potency and tolerability out of over 1540 compounds.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises at least one modified internucleoside linkage, at least onemodified sugar, and/or at least one modified nucleobase.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises at least one modified sugar. In certain embodiments, at leastone modified sugar comprises a 2′-O-methoxyethyl group. In certainembodiments, at least one modified sugar is a bicyclic sugar, such as a4′-CH(CH3)-O-2′ group, a 4′-CH2-O-2′ group, or a 4′-(CH2)2-O-2′ group.

In certain embodiments, the modified oligonucleotide comprises at leastone modified internucleoside linkage, such as a phosphorothioateinternucleoside linkage.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing modified oligonucleotidescomprises:

-   -   a gap segment consisting of linked deoxynucleosides;    -   a 5′ wing segment consisting of linked nucleosides; and    -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar. In certain embodiments, the modifiedoligonucleotide is 14 to 80 linked nucleosides in length having anucleobase sequence comprising the sequence recited in any one of SEQ IDNOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. Incertain embodiments, the modified oligonucleotide is 10 to 30 linkednucleosides in length having a nucleobase sequence comprising thesequence recited in any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147,1149, 1016, 955, 1195, and 353. In certain embodiments, the modifiedoligonucleotide is 14 linked nucleosides in length having a nucleobasesequence consisting of the sequence recited in any one of SEQ ID NOs:1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.

In certain embodiments, a compound comprises or consists of a modifiedoligonucleotide 16-80 linked nucleobases in length having a nucleobasesequence comprising the sequence recited in any one of SEQ ID NOs: 1016,1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353, wherein themodified oligonucleotide comprises

-   -   a gap segment consisting of ten linked deoxynucleosides;    -   a 5′ wing segment consisting of three linked nucleosides; and    -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a cEt sugar; wherein each internucleoside linkage is aphosphorothioate linkage and wherein each cytosine is a5-methylcytosine. In certain embodiments, the modified oligonucleotideconsists of 16-30 linked nucleosides. In certain embodiments, themodified oligonucleotide consists of 16 linked nucleosides.

In certain embodiments, a compound comprises or consists of ISIS 863633or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of ISIS 863633or salt thereof, having the following chemical structure:

In certain embodiments, a compound comprises or consists of the sodiumsalt of ISIS 863633, having the following chemical structure:

In any of the foregoing embodiments, the compound or oligonucleotide canbe at least 85%, at least 90%, at least 95%, at least 98%, at least 99%,or 100% complementary to a nucleic acid encoding PCSK9.

In any of the foregoing embodiments, the compound can besingle-stranded. In certain embodiments, the compound comprisesdeoxyribonucleotides. In certain embodiments, the compound isdouble-stranded. In certain embodiments, the compound is double-strandedand comprises ribonucleotides. In any of the foregoing embodiments, thecompound can be an antisense compound or oligomeric compound.

In any of the foregoing embodiments, the compound can be 8 to 80, 10 to30, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linkednucleosides in length. In certain embodiments, the compound comprises orconsists of an oligonucleotide.

In certain embodiments, a compound comprises a modified oligonucleotidedescribed herein and a conjugate group. In certain embodiments, theconjugate group is linked to the modified oligonucleotide at the 5′ endof the modified oligonucleotide. In certain embodiments, the conjugategroup is linked to the modified oligonucleotide at the 3′ end of themodified oligonucleotide. In certain embodiments, the conjugate groupcomprises at least one N-Acetylgalactosamine (GalNAc), at least twoN-Acetylgalactosamines (GalNAcs), or at least threeN-Acetylgalactosamines (GalNAcs).

In certain embodiments, compounds or compositions provided hereincomprise a pharmaceutically acceptable salt of the modifiedoligonucleotide. In certain embodiments, the salt is a sodium salt. Incertain embodiments, the salt is a potassium salt.

In certain embodiments, the compounds or compositions as describedherein are active by virtue of having at least one of an in vitro IC₅₀of less than 5 μM, less than 4.5 μM, less than 4 μM, less than 3.5 μM,less than 3 μM, less than 2.5 μM, less than 2 μM, less than 1.5 μM, lessthan 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, lessthan 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, lessthan 0.2 μM, or less than 0.1 μM.

In certain embodiments, the compounds or compositions as describedherein are highly tolerable as demonstrated by having at least one of anincrease an alanine transaminase (ALT) or aspartate transaminase (AST)value of no more than 4 fold, 3 fold, or 2 fold over saline treatedanimals or an increase in liver, spleen, or kidney weight of no morethan 30%, 20%, 15%, 12%, 10%, 5%, or 2% compared to control treatedanimals.

In certain embodiments, the compounds or compositions as describedherein are highly tolerable as demonstrated by having no increase of ALTor AST over control treated animals. In certain embodiments, thecompounds or compositions as described herein are highly tolerable asdemonstrated by having no increase in liver, spleen, or kidney weightover control animals.

Certain embodiments provide a composition comprising the compound of anyof the aforementioned embodiments or any pharmaceutically acceptablesalt thereof and at least one of a pharmaceutically acceptable carrieror diluent. In certain embodiments, the composition has a viscosity lessthan about 40 centipoise (cP), less than about 30 centipose (cP), lessthan about 20 centipose (cP), less than about 15 centipose (cP), or lessthan about 10 centipose (cP). In certain embodiments, the compositionhaving any of the aforementioned viscosities comprises a compoundprovided herein at a concentration of about 100 mg/mL, about 125 mg/mL,about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL,about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certainembodiments, the composition having any of the aforementionedviscosities and/or compound concentrations has a temperature of roomtemperature or about 20° C., about 21° C., about 22° C., about 23° C.,about 24° C., about 25° C., about 26° C., about 27° C., about 28° C.,about 29° C., or about 30° C.

Certain Indications

Certain embodiments provided herein relate to methods of inhibitingPCSK9 expression, which can be useful for treating, preventing, orameliorating a disease associated with PCSK9 in an individual, byadministration of a compound that targets PCSK9. In certain embodiments,the compound can be a PCSK9 specific inhibitor. In certain embodiments,the compound can be an antisense compound, oligomeric compound, oroligonucleotide targeted to PCSK9.

Examples of diseases associated with PCSK9 treatable, preventable,and/or ameliorable with the methods provided herein include acardiovascular disease, dyslipidemia, mixed dyslipidemia,hypercholesterolemia, a reduction in LDL cholesterol, and reduction inatherogenic apolipoprotein (a) [Lp(a)].

In certain embodiments, a method of treating, preventing, orameliorating a disease associated with PCSK9 in an individual comprisesadministering to the individual a compound comprising a PCSK9 specificinhibitor, thereby treating, preventing, or ameliorating the disease. Incertain embodiments, the individual is identified as having or at riskof having a disease associated with PCSK9. In certain embodiments, thedisease is a cardiovascular disease. In certain embodiments, thecompound comprises an antisense compound targeted to PCSK9. In certainembodiments, the compound comprises an oligonucleotide targeted toPCSK9. In certain embodiments, a compound comprises a modifiedoligonucleotide 8 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 8 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide 14 to 80linked nucleosides in length and having a nucleobase sequence comprisingthe nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide consistingof the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide 14 to 80linked nucleosides in length having a nucleobase sequence comprising anyone of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195,and 353. In certain embodiments, a compound comprises a modifiedoligonucleotide having a nucleobase sequence consisting of any one ofSEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.In any of the foregoing embodiments, the modified oligonucleotide can be10 to 30 linked nucleosides in length. In certain embodiments, thecompound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In anyof the foregoing embodiments, the compound can be single-stranded ordouble-stranded. In any of the foregoing embodiments, the compound canbe an antisense compound or oligomeric compound. In certain embodiments,the compound is administered to the individual parenterally. In certainembodiments, administering the compound reduces LDL-cholesterol levels,reduces Lp(a) levels, induces LDL receptor (LDL-R) activity, andregulates LDL receptor-LDL-cholesterol homeostasis.

In certain embodiments, a method of treating, preventing, orameliorating a cardiovascular disease comprises administering to theindividual a compound comprising a PCSK9 specific inhibitor, therebytreating, preventing, or ameliorating the cardiovascular disease. Incertain embodiments, the cardiovascular disease is dyslipidemia orhypercholesterolemia. In certain embodiments, the compound comprises anantisense compound targeted to PCSK9. In certain embodiments, thecompound comprises an oligonucleotide targeted to PCSK9. In certainembodiments, a compound comprises a modified oligonucleotide 8 to 80linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 3-1540. In certain embodiments, a compound comprises amodified oligonucleotide 14 to 80 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide 14 to 80 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763,1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, acompound comprises a modified oligonucleotide having a nucleobasesequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071,1147, 1149, 1016, 955, 1195, and 353. In any of the foregoingembodiments, the modified oligonucleotide can be 10 to 30 linkednucleosides in length. In certain embodiments, the compound is ISIS863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633,ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally. In certain embodiments,administering the compound reduces LDL-cholesterol levels. In certainembodiments, administering the compound reduces Lp(a) levels. In certainembodiments, administering the compound induces LDL receptor (LDL-R)activity. In certain embodiments, administering the compound regulatesLDL receptor-LDL-cholesterol homeostasis.

In certain embodiments, a method of inhibiting expression of PCSK9 in anindividual having, or at risk of having, a disease associated with PCSK9comprises administering to the individual a compound comprising a PCSK9specific inhibitor, thereby inhibiting expression of PCSK9 in theindividual. In certain embodiments, administering the compound inhibitsexpression of PCSK9 in the liver. In certain embodiments, the disease isa cardiovascular disease. In certain embodiments, the individual has, oris at risk of having dyslipidemia. In certain embodiments, theindividual has, or is at risk of having hypercholesterolemia. In certainembodiments, the individual has, or is at risk of having mixeddyslipidemia. In certain embodiments, the compound comprises anantisense compound targeted to PCSK9. In certain embodiments, thecompound comprises an oligonucleotide targeted to PCSK9. In certainembodiments, a compound comprises a modified oligonucleotide 8 to 80linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 3-1540. In certain embodiments, a compound comprises amodified oligonucleotide 14 to 80 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide 14 to 80 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763,1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, acompound comprises a modified oligonucleotide having a nucleobasesequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071,1147, 1149, 1016, 955, 1195, and 353. In any of the foregoingembodiments, the modified oligonucleotide can be 10 to 30 linkednucleosides in length. In certain embodiments, the compound is ISIS863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633,ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally. In certain embodiments,administering the compound reduces LDL-cholesterol levels. In certainembodiments, administering the compound reduces Lp(a) levels. In certainembodiments, administering the compound induces LDL receptor (LDL-R)activity. In certain embodiments, administering the compound regulatesLDL receptor-LDL-cholesterol homeostasis.

In certain embodiments, a method of inhibiting expression of PCSK9 in acell comprises contacting the cell with a compound comprising a PCSK9specific inhibitor, thereby inhibiting expression of PCSK9 in the cell.In certain embodiments, the cell is a hepatocyte. In certainembodiments, the cell is in the liver. In certain embodiments, the cellis in the liver of an individual who has, or is at risk of having acardiovascular disease. In certain embodiments, the cell is in the liverof an individual who has, or is at risk of having dyslipidemia. Incertain embodiments, the cell is in the liver of an individual who has,or is at risk of having mixed dyslipidemia. In certain embodiments, thecell is in the liver of an individual who has, or is at risk of havinghypercholesterolemia. In certain embodiments, administering the compoundinhibits expression of PCSK9 in the liver. In certain embodiments, theindividual has, or is at risk of having, a cardiovascular disease. Incertain embodiments, the individual has, or is at risk of havingdyslipidemia. In certain embodiments, the individual has, or is at riskof having mixed dyslipidemia. In certain embodiments, the individualhas, or is at risk of having hypercholesterolemia. In certainembodiments, the compound comprises an antisense compound targeted toPCSK9. In certain embodiments, the compound comprises an oligonucleotidetargeted to PCSK9. In certain embodiments, a compound comprises amodified oligonucleotide 8 to 80 linked nucleosides in length and havinga nucleobase sequence comprising at least 8 contiguous nucleobases ofany of the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide 14 to 80linked nucleosides in length and having a nucleobase sequence comprisingthe nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide consistingof the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide 14 to 80linked nucleosides in length having a nucleobase sequence comprising anyone of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195,and 353. In certain embodiments, a compound comprises a modifiedoligonucleotide having a nucleobase sequence consisting of any one ofSEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.In any of the foregoing embodiments, the modified oligonucleotide can be10 to 30 linked nucleosides in length. In certain embodiments, thecompound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In anyof the foregoing embodiments, the compound can be single-stranded ordouble-stranded. In any of the foregoing embodiments, the compound canbe an antisense compound or oligomeric compound.

In certain embodiments, a method of reducing or inhibitingLDL-cholesterol levels, reducing or inhibiting Lp(a) levels, inducingLDL receptor (LDL-R) activity, or regulating LDLreceptor-LDL-cholesterol homeostasis in the liver of an individualhaving, or at risk of having, a disease associated with PCSK9 comprisesadministering to the individual a compound comprising a PCSK9 specificinhibitor, thereby reducing or inhibiting LDL-cholesterol and Lp(a)levels, inducing LDL receptor (LDL-R) activity, and regulating LDLreceptor-LDL-cholesterol homeostasis in the liver of the individual. Incertain embodiments, the individual has, or is at risk of having, acardiovascular disease. In certain embodiments, the individual has, oris at risk of having, dyslipidemia. In certain embodiments, theindividual has, or is at risk of having, mixed dyslipidemia. In certainembodiments, the individual has, or is at risk of having,hypercholesterolemia. In certain embodiments, the compound comprises anantisense compound targeted to PCSK9. In certain embodiments, thecompound comprises an oligonucleotide targeted to PCSK9. In certainembodiments, a compound comprises a modified oligonucleotide 8 to 80linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 3-1540. In certain embodiments, a compound comprises amodified oligonucleotide 14 to 80 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 3-1540.

In certain embodiments, a compound comprises a modified oligonucleotideconsisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540.In certain embodiments, a compound comprises a modified oligonucleotide14 to 80 linked nucleosides in length having a nucleobase sequencecomprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149,1016, 955, 1195, and 353. In certain embodiments, a compound comprises amodified oligonucleotide having a nucleobase sequence consisting of anyone of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195,and 353. In any of the foregoing embodiments, the modifiedoligonucleotide can be 10 to 30 linked nucleosides in length. In certainembodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS863681. In any of the foregoing embodiments, the compound can besingle-stranded or double-stranded. In any of the foregoing embodiments,the compound can be an antisense compound or oligomeric compound. Incertain embodiments, the compound is administered to the individualparenterally. In certain embodiments, the individual is identified ashaving or at risk of having a disease associated with PCSK9.

Certain embodiments are drawn to a compound comprising a PCSK9 specificinhibitor for use in treating a disease associated with PCSK9. Incertain embodiments, the disease is a cardiovascular disease. In certainembodiments, the disease is dyslipidemia. In certain embodiments, thedisease is mixed dyslipidemia. In certain embodiments, the disease ishypercholesterolemia In certain embodiments, the compound comprises anantisense compound targeted to PCSK9. In certain embodiments, thecompound comprises an oligonucleotide targeted to PCSK9. In certainembodiments, a compound comprises a modified oligonucleotide 8 to 80linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 3-1540. In certain embodiments, a compound comprises amodified oligonucleotide 14 to 80 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide 14 to 80 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763,1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, acompound comprises a modified oligonucleotide having a nucleobasesequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071,1147, 1149, 1016, 955, 1195, and 353. In any of the foregoingembodiments, the modified oligonucleotide can be 10 to 30 linkednucleosides in length. In certain embodiments, the compound is ISIS863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633,ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound. In certain embodiments, the compound isadministered to the individual parenterally.

Certain embodiments are drawn to a compound comprising a PCSK9 specificinhibitor for use in reducing or inhibiting LDL-cholesterol, reducing orinhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, andregulating LDL receptor-LDL-cholesterol homeostasis of an individualhaving or at risk of having a cardiovascular disease. In certainembodiments, the cardiovascular disease is dyslipidemia orhypercholesterolemia. In certain embodiments, the compound comprises anantisense compound targeted to PCSK9. In certain embodiments, thecompound comprises an oligonucleotide targeted to PCSK9. In certainembodiments, a compound comprises a modified oligonucleotide 8 to 80linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 3-1540. In certain embodiments, a compound comprises amodified oligonucleotide 14 to 80 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide 14 to 80 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763,1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, acompound comprises a modified oligonucleotide having a nucleobasesequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071,1147, 1149, 1016, 955, 1195, and 353. In any of the foregoingembodiments, the modified oligonucleotide can be 10 to 30 linkednucleosides in length. In certain embodiments, the compound is ISIS863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633,ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound.

Certain embodiments are drawn to use of a compound comprising a PCSK9specific inhibitor for the manufacture or preparation of a medicamentfor treating a disease associated with PCSK9. Certain embodiments aredrawn to use of a compound comprising a PCSK9 specific inhibitor for thepreparation of a medicament for treating a disease associated withPCSK9. In certain embodiments, the disease is a cardiovascular disease.In certain embodiments, the disease is dyslipidemia. In certainembodiments, the disease is mixed dyslipidemia. In certain embodiments,the disease is hypercholesterolemia. In certain embodiments, thecompound comprises an antisense compound targeted to PCSK9. In certainembodiments, the compound comprises an oligonucleotide targeted toPCSK9. In certain embodiments, a compound comprises a modifiedoligonucleotide 8 to 80 linked nucleosides in length and having anucleobase sequence comprising at least 8 contiguous nucleobases of anyof the nucleobase sequences of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide 14 to 80linked nucleosides in length and having a nucleobase sequence comprisingthe nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide consistingof the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certainembodiments, a compound comprises a modified oligonucleotide 14 to 80linked nucleosides in length having a nucleobase sequence comprising anyone of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195,and 353. In certain embodiments, a compound comprises a modifiedoligonucleotide having a nucleobase sequence consisting of any one ofSEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.In any of the foregoing embodiments, the modified oligonucleotide can be10 to 30 linked nucleosides in length. In certain embodiments, thecompound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In anyof the foregoing embodiments, the compound can be single-stranded ordouble-stranded. In any of the foregoing embodiments, the compound canbe an antisense compound or oligomeric compound.

Certain embodiments are drawn to use of a compound comprising a PCSK9specific inhibitor for the manufacture or preparation of a medicamentfor reducing or inhibiting LDL-cholesterol levels, reducing orinhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, andregulating LDL receptor-LDL-cholesterol homeostasis of an individualhaving or at risk of having a cardiovascular disease. In certainembodiments, the cardiovascular disease is dyslipidemia orhypercholesterolemia. Certain embodiments are drawn to use of a compoundcomprising a PCSK9 specific inhibitor for the preparation of amedicament for reducing or inhibiting LDL-cholesterol levels, reducingor inhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, andregulating LDL receptor-LDL-cholesterol homeostasis of an individualhaving or at risk of having a cardiovascular disease. In certainembodiments, the cardiovascular disease is dyslipidemia orhypercholesterolemia. In certain embodiments, the compound comprises anantisense compound targeted to PCSK9. In certain embodiments, thecompound comprises an oligonucleotide targeted to PCSK9. In certainembodiments, a compound comprises a modified oligonucleotide 8 to 80linked nucleosides in length and having a nucleobase sequence comprisingat least 8 contiguous nucleobases of any of the nucleobase sequences ofSEQ ID NOs: 3-1540. In certain embodiments, a compound comprises amodified oligonucleotide 14 to 80 linked nucleosides in length andhaving a nucleobase sequence comprising the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide consisting of the nucleobase sequence of anyone of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprisesa modified oligonucleotide 14 to 80 linked nucleosides in length havinga nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763,1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, acompound comprises a modified oligonucleotide having a nucleobasesequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071,1147, 1149, 1016, 955, 1195, and 353. In any of the foregoingembodiments, the modified oligonucleotide can be 10 to 30 linkednucleosides in length. In certain embodiments, the compound is ISIS863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633,ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoingembodiments, the compound can be single-stranded or double-stranded. Inany of the foregoing embodiments, the compound can be an antisensecompound or oligomeric compound.

In any of the foregoing methods or uses, the compound can be targeted toPCSK9. In certain embodiments, the compound comprises or consists of amodified oligonucleotide, for example a modified oligonucleotide 8 to 80linked nucleosides in length, 10 to 30 linked nucleosides in length, 12to 30 linked nucleosides in length, or 20 linked nucleosides in length.In certain embodiments, the modified oligonucleotide is at least 80%,85%, 90%, 95% or 100% complementary to any of the nucleobase sequencesrecited in SEQ ID NOs: 1, 2, 1545-1550. In certain embodiments, themodified oligonucleotide comprises at least one modified internucleosidelinkage, at least one modified sugar and/or at least one modifiednucleobase. In certain embodiments, the modified internucleoside linkageis a phosphorothioate internucleoside linkage, the modified sugar is abicyclic sugar or a 2′-O-methoxyethyl, and the modified nucleobase is a5-methylcytosine. In certain embodiments, the modified oligonucleotidecomprises a gap segment consisting of linked deoxynucleosides; a 5′ wingsegment consisting of linked nucleosides; and a 3′ wing segmentconsisting of linked nucleosides, wherein the gap segment is positionedimmediately adjacent to and between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises amodified sugar.

In any of the foregoing embodiments, the modified oligonucleotide is 12to 30, 15 to 30, 15 to 25, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to24, 20 to 24, 19 to 22, 20 to 22, 16 to 20, or 17 or 20 linkednucleosides in length. In certain embodiments, the modifiedoligonucleotide is at least 80%, 85%, 90%, 95% or 100% complementary toany of the nucleobase sequences recited in SEQ ID NOs: 1, 2, 1545-1550.In certain embodiments, the modified oligonucleotide comprises at leastone modified internucleoside linkage, at least one modified sugar and/orat least one modified nucleobase. In certain embodiments, the modifiedinternucleoside linkage is a phosphorothioate internucleoside linkage,the modified sugar is a bicyclic sugar or a 2′-O-methoxyethyl, and themodified nucleobase is a 5-methylcytosine. In certain embodiments, themodified oligonucleotide comprises a gap segment consisting of linked2′-deoxynucleosides; a 5′ wing segment consisting of linked nucleosides;and a 3′ wing segment consisting of linked nucleosides, wherein the gapsegment is positioned immediately adjacent to and between the 5′ wingsegment and the 3′ wing segment and wherein each nucleoside of each wingsegment comprises a modified sugar.

In any of the foregoing methods or uses, the compound comprises orconsists of a modified oligonucleotide 14 to 30 linked nucleosides inlength and having a nucleobase sequence comprising any one of SEQ IDNOs: 3-1540, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked 2′-deoxynucleosides;    -   a 5′ wing segment consisting of linked nucleosides; and    -   a 3′ wing segment consisting of linked nucleosides;        wherein the gap segment is positioned between the 5′ wing        segment and the 3′ wing segment and wherein each nucleoside of        each wing segment comprises a modified sugar.

In any of the foregoing methods or uses, the compound comprises orconsists a modified oligonucleotide 16 to 80 linked nucleobases inlength having a nucleobase sequence comprising or consisting of thesequence recited in any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147,1149, 1016, 955, 1195, and 353, wherein the modified oligonucleotidecomprises

-   -   a gap segment consisting of ten linked deoxynucleosides;    -   a 5′ wing segment consisting of three linked nucleosides; and    -   a 3′ wing segment consisting of three linked nucleosides;        -   wherein the gap segment is positioned between the 5′ wing            segment and the 3′ wing segment, wherein each nucleoside of            each wing segment comprises a cEt sugar; wherein each            internucleoside linkage is a phosphorothioate linkage;            wherein each cytosine is a 5-methylcytosine. In certain            embodiments, the modified oligonucleotide is 16-80 linked            nucleosides in length. In certain embodiments, the modified            oligonucleotide is 16-30 linked nucleosides in length.

In any of the foregoing methods or uses, the compound comprises orconsists of ISIS 863633 or salt thereof, having the following chemicalstructure:

In any of the foregoing methods or uses, the compound comprises orconsists of ISIS 863633 or salt thereof, having the following chemicalstructure:

In any of the foregoing methods or uses, the compound comprises orconsists of the sodium salt of ISIS 863633, having the followingchemical structure:

In any of the foregoing methods or uses, the compound can beadministered parenterally. For example, in certain embodiments thecompound can be administered through injection or infusion. Parenteraladministration includes subcutaneous administration, intravenousadministration, intramuscular administration, intraarterialadministration, intraperitoneal administration, or intracranialadministration, e.g. intrathecal or intracerebroventricularadministration.

Certain Compounds

In certain embodiments, compounds described herein can be antisensecompounds. In certain embodiments, the antisense compound comprises orconsists of an oligomeric compound. In certain embodiments, theoligomeric compound comprises a modified oligonucleotide. In certainembodiments, the modified oligonucleotide has a nucleobase sequencecomplementary to that of a target nucleic acid.

In certain embodiments, a compound described herein comprises orconsists of a modified oligonucleotide. In certain embodiments, themodified oligonucleotide has a nucleobase sequence complementary to thatof a target nucleic acid.

In certain embodiments, a compound or antisense compound issingle-stranded. Such a single-stranded compound or antisense compoundcomprises or consists of an oligomeric compound. In certain embodiments,such an oligomeric compound comprises or consists of an oligonucleotideand optionally a conjugate group. In certain embodiments, theoligonucleotide is an antisense oligonucleotide. In certain embodiments,the oligonucleotide is modified. In certain embodiments, theoligonucleotide of a single-stranded antisense compound or oligomericcompound comprises a self-complementary nucleobase sequence.

In certain embodiments, compounds are double-stranded. Suchdouble-stranded compounds comprise a first modified oligonucleotidehaving a region complementary to a target nucleic acid and a secondmodified oligonucleotide having a region complementary to the firstmodified oligonucleotide. In certain embodiments, the modifiedoligonucleotide is an RNA oligonucleotide. In such embodiments, thethymine nucleobase in the modified oligonucleotide is replaced by auracil nucleobase. In certain embodiments, compound comprises aconjugate group. In certain embodiments, one of the modifiedoligonucleotides is conjugated. In certain embodiments, both themodified oligonucleotides are conjugated. In certain embodiments, thefirst modified oligonucleotide is conjugated. In certain embodiments,the second modified oligonucleotide is conjugated. In certainembodiments, the first modified oligonucleotide is 12-30 linkednucleosides in length and the second modified oligonucleotide is 12-30linked nucleosides in length. In certain embodiments, one of themodified oligonucleotides has a nucleobase sequence comprising at least8 contiguous nucleobases of any of SEQ ID NOs: 3-1540.

In certain embodiments, antisense compounds are double-stranded. Suchdouble-stranded antisense compounds comprise a first oligomeric compoundhaving a region complementary to a target nucleic acid and a secondoligomeric compound having a region complementary to the firstoligomeric compound. The first oligomeric compound of such doublestranded antisense compounds typically comprises or consists of amodified oligonucleotide and optionally a conjugate group. Theoligonucleotide of the second oligomeric compound of suchdouble-stranded antisense compound may be modified or unmodified. Eitheror both oligomeric compounds of a double-stranded antisense compound maycomprise a conjugate group. The oligomeric compounds of double-strandedantisense compounds may include non-complementary overhangingnucleosides.

Examples of single-stranded and double-stranded compounds include butare not limited to oligonucleotides, siRNAs, microRNA targetingoligonucleotides, and single-stranded RNAi compounds, such as smallhairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNAmimics.

In certain embodiments, a compound described herein has a nucleobasesequence that, when written in the 5′ to 3′ direction, comprises thereverse complement of the target segment of a target nucleic acid towhich it is targeted.

In certain embodiments, a compound described herein comprises anoligonucleotide 10 to 30 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 12to 30 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 12 to 22 linked subunitsin length. In certain embodiments, compound described herein comprisesan oligonucleotide 14 to 30 linked subunits in length. In certainembodiments, compound described herein comprises an oligonucleotide 14to 20 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 15 to 30 linked subunitsin length. In certain embodiments, a compound described herein comprisesan oligonucleotide 15 to 20 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 16to 30 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 16 to 20 linked subunitsin length. In certain embodiments, a compound described herein comprisesan oligonucleotide 17 to 30 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 17to 20 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 18 to 30 linked subunitsin length. In certain embodiments, a compound described herein comprisesan oligonucleotide 18 to 21 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 18to 20 linked subunits in length. In certain embodiments, a compounddescribed herein comprises an oligonucleotide 20 to 30 linked subunitsin length. In other words, such oligonucleotides are 12 to 30 linkedsubunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits inlength, respectively. In certain embodiments, a compound describedherein comprises an oligonucleotide 14 linked subunits in length. Incertain embodiments, a compound described herein comprises anoligonucleotide 16 linked subunits in length. In certain embodiments, acompound described herein comprises an oligonucleotide 17 linkedsubunits in length. In certain embodiments, compound described hereincomprises an oligonucleotide 18 linked subunits in length. In certainembodiments, a compound described herein comprises an oligonucleotide 19linked subunits in length. In certain embodiments, a compound describedherein comprises an oligonucleotide 20 linked subunits in length. Inother embodiments, a compound described herein comprises anoligonucleotide 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to30 linked subunits. In certain such embodiments, the compound describedherein comprises an oligonucleotide 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, ora range defined by any two of the above values. In some embodiments thelinked subunits are nucleotides, nucleosides, or nucleobases.

In certain embodiments, the compound may further comprise additionalfeatures or elements, such as a conjugate group, that are attached tothe oligonucleotide. In certain embodiments, such compounds areantisense compounds. In certain embodiments, such compounds areoligomeric compounds. In embodiments where a conjugate group comprises anucleoside (i.e. a nucleoside that links the conjugate group to theoligonucleotide), the nucleoside of the conjugate group is not countedin the length of the oligonucleotide.

In certain embodiments, compounds may be shortened or truncated. Forexample, a single subunit may be deleted from the 5′ end (5′truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated compound targeted to a PCSK9 nucleic acid mayhave two subunits deleted from the 5′ end, or alternatively may have twosubunits deleted from the 3′ end, of the compound. Alternatively, thedeleted nucleosides may be dispersed throughout the compound.

When a single additional subunit is present in a lengthened compound,the additional subunit may be located at the 5′ or 3′ end of thecompound. When two or more additional subunits are present, the addedsubunits may be adjacent to each other, for example, in a compoundhaving two subunits added to the 5′ end (5′ addition), or alternativelyto the 3′ end (3′ addition), of the compound. Alternatively, the addedsubunits may be dispersed throughout the compound.

It is possible to increase or decrease the length of a compound, such asan oligonucleotide, and/or introduce mismatch bases without eliminatingactivity (Woolf et al. Proc. Natl. Acad. Sci. USA 1992, 89:7305-7309;Gautschi et al. J. Natl. Cancer Inst. March 2001, 93:463-471; Maher andDolnick Nuc. Acid. Res. 1998, 16:3341-3358). However, seemingly smallchanges in oligonucleotide sequence, chemistry and motif can make largedifferences in one or more of the many properties required for clinicaldevelopment (Seth et al. J. Med. Chem. 2009, 52, 10; Egli et al. J. Am.Chem. Soc. 2011, 133, 16642).

In certain embodiments, compounds described herein are interfering RNAcompounds (RNAi), which include double-stranded RNA compounds (alsoreferred to as short-interfering RNA or siRNA) and single-stranded RNAicompounds (or ssRNA). Such compounds work at least in part through theRISC pathway to degrade and/or sequester a target nucleic acid (thus,include microRNA/microRNA-mimic compounds). As used herein, the termsiRNA is meant to be equivalent to other terms used to describe nucleicacid molecules that are capable of mediating sequence specific RNAi, forexample short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, short interferingmodified oligonucleotide, chemically modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term “RNAi” is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics.

In certain embodiments, a compound described herein can comprise any ofthe oligonucleotide sequences targeted to PCSK9 described herein. Incertain embodiments, the compound can be double-stranded. In certainembodiments, the compound comprises a first strand comprising at leastan 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguousnucleobase portion of any one of SEQ ID NOs: 3-1540 and a second strand.In certain embodiments, the compound comprises a first strand comprisingthe nucleobase sequence of any one of SEQ ID NOs: 3-1540 and a secondstrand. In certain embodiments, the compound comprises ribonucleotidesin which the first strand has uracil (U) in place of thymine (T) in anyone of SEQ ID NOs: 3-1540. In certain embodiments, the compoundcomprises (i) a first strand comprising a nucleobase sequencecomplementary to the site on PCSK9 to which any of SEQ ID NOs: 3-1540 istargeted, and (ii) a second strand. In certain embodiments, the compoundcomprises one or more modified nucleotides in which the 2′ position inthe sugar contains a halogen (such as fluorine group; 2′-F) or containsan alkoxy group (such as a methoxy group; 2′-OMe). In certainembodiments, the compound comprises at least one 2′-F sugar modificationand at least one 2′-OMe sugar modification. In certain embodiments, theat least one 2′-F sugar modification and at least one 2′-OMe sugarmodification are arranged in an alternating pattern for at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20contiguous nucleobases along a strand of the dsRNA compound. In certainembodiments, the compound comprises one or more linkages betweenadjacent nucleotides other than a naturally-occurring phosphodiesterlinkage. Examples of such linkages include phosphoramide,phosphorothioate, and phosphorodithioate linkages. The compounds mayalso be chemically modified nucleic acid molecules as taught in U.S.Pat. No. 6,673,661. In other embodiments, the compound contains one ortwo capped strands, as disclosed, for example, by WO 00/63364, filedApr. 19, 2000.

In certain embodiments, the first strand of the compound is an siRNAguide strand and the second strand of the compound is an siRNA passengerstrand. In certain embodiments, the second strand of the compound iscomplementary to the first strand. In certain embodiments, each strandof the compound is 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosidesin length. In certain embodiments, the first or second strand of thecompound can comprise a conjugate group.

In certain embodiments, a compound described herein can comprise any ofthe oligonucleotide sequences targeted to PCSK9 described herein. Incertain embodiments, the compound is single stranded. In certainembodiments, such a compound is a single-stranded RNAi (ssRNAi)compound. In certain embodiments, the compound comprises at least an 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobaseportion of any one of SEQ ID NOs: 3-1540. In certain embodiments, thecompound comprises the nucleobase sequence of any one of SEQ ID NOs:3-1540 In certain embodiments, the compound comprises ribonucleotides inwhich uracil (U) is in place of thymine (T) in any one of SEQ ID NOs:3-1540. In certain embodiments, the compound comprises a nucleobasesequence complementary to the site on PCSK9 to which any of SEQ ID NOs:3-1540 is targeted. In certain embodiments, the compound comprises oneor more modified nucleotides in which the 2′ position in the sugarcontains a halogen (such as fluorine group; 2′-F) or contains an alkoxygroup (such as a methoxy group; 2′-OMe). In certain embodiments, thecompound comprises at least one 2′-F sugar modification and at least one2′-OMe sugar modification. In certain embodiments, the at least one 2′-Fsugar modification and at least one 2′-OMe sugar modification arearranged in an alternating pattern for at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobasesalong a strand of the compound. In certain embodiments, the compoundcomprises one or more linkages between adjacent nucleotides other than anaturally-occurring phosphodiester linkage. Examples of such linkagesinclude phosphoramide, phosphorothioate, and phosphorodithioatelinkages. The compounds may also be chemically modified nucleic acidmolecules as taught in U.S. Pat. No. 6,673,661. In other embodiments,the compound contains a capped strand, as disclosed, for example, by WO00/63364, filed Apr. 19, 2000. In certain embodiments, the compoundconsists of 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides. Incertain embodiments, the compound can comprise a conjugate group.

In certain embodiments, compounds described herein comprise modifiedoligonucleotides. Certain modified oligonucleotides have one or moreasymmetric center and thus give rise to enantiomers, diastereomers, andother stereoisomeric configurations that may be defined, in terms ofabsolute stereochemistry, as (R) or (S), as a or f such as for sugaranomers, or as (D) or (L) such as for amino acids etc. Included in themodified oligonucleotides provided herein are all such possible isomers,including their racemic and optically pure forms, unless specifiedotherwise. Likewise, all cis- and trans-isomers and tautomeric forms arealso included.

The compounds described herein include variations in which one or moreatoms are replaced with a non-radioactive isotope or radioactive isotopeof the indicated element. For example, compounds herein that comprisehydrogen atoms encompass all possible deuterium substitutions for eachof the ¹H hydrogen atoms. Isotopic substitutions encompassed by thecompounds herein include but are not limited to: ²H or ³H in place of¹H, ¹³C or ¹⁴C in place of ¹²C, ¹⁵N in place of ¹⁴N, ¹⁷O or ¹⁸O in placeof ¹⁶O, and ³³S, ³⁴S, ³⁵S, or ³⁶S in place of ³²S. In certainembodiments, non-radioactive isotopic substitutions may impart newproperties on the compound that are beneficial for use as a therapeuticor research tool. In certain embodiments, radioactive isotopicsubstitutions may make the compound suitable for research or diagnosticpurposes, such as an imaging assay.

Certain Mechanisms

In certain embodiments, compounds described herein comprise or consistof modified oligonucleotides. In certain embodiments, compoundsdescribed herein are antisense compounds. In certain embodiments,compounds comprise oligomeric compounds. In certain embodiments,compounds described herein are capable of hybridizing to a targetnucleic acid, resulting in at least one antisense activity. In certainembodiments, compounds described herein selectively affect one or moretarget nucleic acid. Such compounds comprise a nucleobase sequence thathybridizes to one or more target nucleic acid, resulting in one or moredesired antisense activity and does not hybridize to one or morenon-target nucleic acid or does not hybridize to one or more non-targetnucleic acid in such a way that results in a significant undesiredantisense activity.

In certain antisense activities, hybridization of a compound describedherein to a target nucleic acid results in recruitment of a protein thatcleaves the target nucleic acid. For example, certain compoundsdescribed herein result in RNase H mediated cleavage of the targetnucleic acid. RNase H is a cellular endonuclease that cleaves the RNAstrand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need notbe unmodified DNA. In certain embodiments, compounds described hereinare sufficiently “DNA-like”to elicit RNase H activity. Further, incertain embodiments, one or more non-DNA-like nucleoside in the gap of agapmer is tolerated.

In certain antisense activities, compounds described herein or a portionof the compound is loaded into an RNA-induced silencing complex (RISC),ultimately resulting in cleavage of the target nucleic acid. Forexample, certain compounds described herein result in cleavage of thetarget nucleic acid by Argonaute. Compounds that are loaded into RISCare RNAi compounds. RNAi compounds may be double-stranded (siRNA) orsingle-stranded (ssRNA).

In certain embodiments, hybridization of compounds described herein to atarget nucleic acid does not result in recruitment of a protein thatcleaves that target nucleic acid. In certain such embodiments,hybridization of the compound to the target nucleic acid results inalteration of splicing of the target nucleic acid. In certainembodiments, hybridization of the compound to a target nucleic acidresults in inhibition of a binding interaction between the targetnucleic acid and a protein or other nucleic acid. In certain suchembodiments, hybridization of the compound to a target nucleic acidresults in alteration of translation of the target nucleic acid.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid, a change in theratio of splice variants of a nucleic acid or protein, and/or aphenotypic change in a cell or animal.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, compounds described herein comprise or consistof an oligonucleotide comprising a region that is complementary to atarget nucleic acid. In certain embodiments, the target nucleic acid isan endogenous RNA molecule. In certain embodiments, the target nucleicacid encodes a protein. In certain such embodiments, the target nucleicacid is selected from: an mRNA and a pre-mRNA, including intronic,exonic and untranslated regions. In certain embodiments, the target RNAis an mRNA. In certain embodiments, the target nucleic acid is apre-mRNA. In certain such embodiments, the target region is entirelywithin an intron. In certain embodiments, the target region spans anintron/exon junction. In certain embodiments, the target region is atleast 50% within an intron.

Nucleotide sequences that encode PCSK9 include, without limitation, thefollowing: RefSEQ No. GENBANK Accession No. NM_174936.3 (SEQ ID NO: 1),GENBANK Accession No. NC_000001.11 truncated from nucleotides 55036001to 55068000 (SEQ ID NO: 2), GENBANK Accession No. AK124635.1 (SEQ ID NO:1545), GENBANK Accession No. NT_032977.8 truncated from nucleotides25475000 to 25504000 (SEQ ID NO: 1546), GENBANK Accession No. DA092236.1(SEQ ID NO: 1547), GENBANK Accession No. DA803830.1 (SEQ ID NO: 1548),GENBANK Accession No. DC352135.1 (SEQ ID NO: 1549), and GENBANKAccession No. NR_110451.1 (SEQ ID NO: 1550).

Hybridization

In some embodiments, hybridization occurs between a compound disclosedherein and a PCSK9 nucleic acid. The most common mechanism ofhybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Hybridizationconditions are sequence-dependent and are determined by the nature andcomposition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the compounds provided herein are specifically hybridizablewith a PCSK9 nucleic acid.

Complementarity

An oligonucleotide is said to be complementary to another nucleic acidwhen the nucleobase sequence of such oligonucleotide or one or moreregions thereof matches the nucleobase sequence of anotheroligonucleotide or nucleic acid or one or more regions thereof when thetwo nucleobase sequences are aligned in opposing directions. Nucleobasematches or complementary nucleobases, as described herein, are limitedto the following pairs: adenine (A) and thymine (T), adenine (A) anduracil (U), cytosine (C) and guanine (G), and 5-methyl cytosine (mC) andguanine (G) unless otherwise specified. Complementary oligonucleotidesand/or nucleic acids need not have nucleobase complementarity at eachnucleoside and may include one or more nucleobase mismatches. Anoligonucleotide is fully complementary or 100% complementary when sucholigonucleotides have nucleobase matches at each nucleoside without anynucleobase mismatches.

In certain embodiments, compounds described herein comprise or consistof modified oligonucleotides. In certain embodiments, compoundsdescribed herein are antisense compounds. In certain embodiments,compounds comprise oligomeric compounds. Non-complementary nucleobasesbetween a compound and a PCSK9 nucleic acid may be tolerated providedthat the compound remains able to specifically hybridize to a targetnucleic acid. Moreover, a compound may hybridize over one or moresegments of a PCSK9 nucleic acid such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure, mismatch or hairpin structure).

In certain embodiments, the compounds provided herein, or a specifiedportion thereof, are, are at least, or are up to 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a PCSK9 nucleic acid, a target region, target segment,or specified portion thereof. In certain embodiments, the compoundsprovided herein, or a specified portion thereof, are 70% to 75%, 75% to80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, or any number inbetween these ranges, complementary to a PCSK9 nucleic acid, a targetregion, target segment, or specified portion thereof. Percentcomplementarity of a compound with a target nucleic acid can bedetermined using routine methods.

For example, a compound in which 18 of 20 nucleobases of the compoundare complementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity. In this example,the remaining non-complementary nucleobases may be clustered orinterspersed with complementary nucleobases and need not be contiguousto each other or to complementary nucleobases. As such, a compound whichis 18 nucleobases in length having four non-complementary nucleobaseswhich are flanked by two regions of complete complementarity with thetarget nucleic acid would have 77.8% overall complementarity with thetarget nucleic acid. Percent complementarity of a compound with a regionof a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology,sequence identity or complementarity, can be determined by, for example,the Gap program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, Madison Wis.),using default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, compounds described herein, or specifiedportions thereof, are fully complementary (i.e. 100% complementary) to atarget nucleic acid, or specified portion thereof. For example, acompound may be fully complementary to a PCSK9 nucleic acid, or a targetregion, or a target segment or target sequence thereof. As used herein,“fully complementary” means each nucleobase of a compound iscomplementary to the corresponding nucleobase of a target nucleic acid.For example, a 20 nucleobase compound is fully complementary to a targetsequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the compound. Fully complementary can also beused in reference to a specified portion of the first and/or the secondnucleic acid. For example, a 20 nucleobase portion of a 30 nucleobasecompound can be “fully complementary” to a target sequence that is 400nucleobases long. The 20 nucleobase portion of the 30 nucleobasecompound is fully complementary to the target sequence if the targetsequence has a corresponding 20 nucleobase portion wherein eachnucleobase is complementary to the 20 nucleobase portion of thecompound. At the same time, the entire 30 nucleobase compound may or maynot be fully complementary to the target sequence, depending on whetherthe remaining 10 nucleobases of the compound are also complementary tothe target sequence.

In certain embodiments, compounds described herein comprise one or moremismatched nucleobases relative to the target nucleic acid. In certainsuch embodiments, antisense activity against the target is reduced bysuch mismatch, but activity against a non-target is reduced by a greateramount. Thus, in certain such embodiments selectivity of the compound isimproved. In certain embodiments, the mismatch is specificallypositioned within an oligonucleotide having a gapmer motif In certainsuch embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8from the 5′-end of the gap region. In certain such embodiments, themismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3′-end of thegap region. In certain such embodiments, the mismatch is at position 1,2, 3, or 4 from the 5′-end of the wing region. In certain suchembodiments, the mismatch is at position 4, 3, 2, or 1 from the 3′-endof the wing region. In certain embodiments, the mismatch is specificallypositioned within an oligonucleotide not having a gapmer motif Incertain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 from the 5′-end of the oligonucleotide. Incertain such embodiments, the mismatch is at position, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 from the 3′-end of the oligonucleotide.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the compound. Alternatively, the non-complementary nucleobaseor nucleobases may be at an internal position of the compound. When twoor more non-complementary nucleobases are present, they may becontiguous (i.e. linked) or non-contiguous. In one embodiment, anon-complementary nucleobase is located in the wing segment of a gapmeroligonucleotide.

In certain embodiments, compounds described herein that are, or are upto 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in lengthcomprise no more than 4, no more than 3, no more than 2, or no more than1 non-complementary nucleobase(s) relative to a target nucleic acid,such as a PCSK9 nucleic acid, or specified portion thereof.

In certain embodiments, compounds described herein that are, or are upto 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleobases in length comprise no more than 6, no morethan 5, no more than 4, no more than 3, no more than 2, or no more than1 non-complementary nucleobase(s) relative to a target nucleic acid,such as a PCSK9 nucleic acid, or specified portion thereof.

In certain embodiments, compounds described herein also include thosewhich are complementary to a portion of a target nucleic acid. As usedherein, “portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof a compound. In certain embodiments, the—compounds, are complementaryto at least an 8 nucleobase portion of a target segment. In certainembodiments, the compounds are complementary to at least a 9 nucleobaseportion of a target segment. In certain embodiments, the compounds arecomplementary to at least a 10 nucleobase portion of a target segment.In certain embodiments, the compounds are complementary to at least an11 nucleobase portion of a target segment. In certain embodiments, thecompounds are complementary to at least a 12 nucleobase portion of atarget segment. In certain embodiments, the compounds are complementaryto at least a 13 nucleobase portion of a target segment. In certainembodiments, the compounds are complementary to at least a 14 nucleobaseportion of a target segment. In certain embodiments, the compounds arecomplementary to at least a 15 nucleobase portion of a target segment.In certain embodiments, the compounds are complementary to at least a 16nucleobase portion of a target segment. Also contemplated are compoundsthat are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more nucleobase portion of a target segment, or a rangedefined by any two of these values.

Identity

The compounds provided herein may also have a defined percent identityto a particular nucleotide sequence, SEQ ID NO, or compound representedby a specific ION number, or portion thereof. In certain embodiments,compounds described herein are antisense compounds or oligomericcompounds. In certain embodiments, compounds described herein aremodified oligonucleotides. As used herein, a compound is identical tothe sequence disclosed herein if it has the same nucleobase pairingability. For example, a RNA which contains uracil in place of thymidinein a disclosed DNA sequence would be considered identical to the DNAsequence since both uracil and thymidine pair with adenine. Shortenedand lengthened versions of the compounds described herein as well ascompounds having non-identical bases relative to the compounds providedherein also are contemplated. The non-identical bases may be adjacent toeach other or dispersed throughout the compound. Percent identity of ancompound is calculated according to the number of bases that haveidentical base pairing relative to the sequence to which it is beingcompared.

In certain embodiments, compounds described herein, or portions thereof,are, or are at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to one or more of the compounds orSEQ ID NOs, or a portion thereof, disclosed herein. In certainembodiments, compounds described herein are about 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or anypercentage between such values, to a particular nucleotide sequence, SEQID NO, or compound represented by a specific ION number, or portionthereof, in which the compounds comprise an oligonucleotide having oneor more mismatched nucleobases. In certain such embodiments, themismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fromthe 5′-end of the oligonucleotide. In certain such embodiments, themismatch is at position, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the3′-end of the oligonucleotide.

In certain embodiments, compounds described herein comprise or consistof antisense compounds. In certain embodiments, a portion of theantisense compound is compared to an equal length portion of the targetnucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is comparedto an equal length portion of the target nucleic acid.

In certain embodiments, compounds described herein comprise or consistof oligonucleotides. In certain embodiments, a portion of theoligonucleotide is compared to an equal length portion of the targetnucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is comparedto an equal length portion of the target nucleic acid.

Certain Modified Compounds

In certain embodiments, compounds described herein comprise or consistof oligonucleotides consisting of linked nucleosides. Oligonucleotidesmay be unmodified oligonucleotides (RNA or DNA) or may be modifiedoligonucleotides. Modified oligonucleotides comprise at least onemodification relative to unmodified RNA or DNA (i.e., comprise at leastone modified nucleoside (comprising a modified sugar moiety and/or amodified nucleobase) and/or at least one modified internucleosidelinkage).

A. Modified Nucleosides Modified nucleosides comprise a modified sugarmoiety or a modified nucleobase or both a modified sugar moiety and amodified nucleobase.

1. Modified Sugar Moieties

In certain embodiments, sugar moieties are non-bicyclic modified sugarmoieties. In certain embodiments, modified sugar moieties are bicyclicor tricyclic sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of other types of modifiedsugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties comprising a furanosyl ring with one or moreacyclic substituent, including but not limited to substituents at the2′, 4′, and/or 5′ positions. In certain embodiments one or more acyclicsubstituent of non-bicyclic modified sugar moieties is branched.Examples of 2′-substituent groups suitable for non-bicyclic modifiedsugar moieties include but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or“O-methyl”), and 2′-O(CH₂)₂OCH₃, (“MOE”). In certain embodiments,2′-substituent groups are selected from among: halo, allyl, amino,azido, SH, CN, OCN, CF₃, OCF₃, O—C₁-C₁₀ alkoxy, O—C₁-C₁₀ substitutedalkoxy, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl, S-alkyl,N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl, O-alkynyl,S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O(CH₂)₂ON(R_(m))(R_(n)) orOCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently,H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat.No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al.,U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from among: hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy, thioalkyl, halogen,alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groupssuitable for linearly non-bicyclic modified sugar moieties include butare not limited to alkoxy (e.g., methoxy), alkyl, and those described inManoharan et al., WO 2015/106128. Examples of 5′-substituent groupssuitable for non-bicyclic modified sugar moieties include but are notlimited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certainembodiments, non-bicyclic modified sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties and the modified sugar moieties and modified nucleosidesdescribed in Migawa et al., US2010/190837 and Rajeev et al.,US2013/0203836.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a linear2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂,CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O(CH₂)₂ON(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m) and R_(n) is,independently, H, an amino protecting group, or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a linear2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃,O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, andOCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a linear2′-substituent group selected from: F, OCH₃, and OCH₂CH₂OCH₃.

Nucleosides comprising modified sugar moieties, such as non-bicyclicmodified sugar moieties, are referred to by the position(s) of thesubstitution(s) on the sugar moiety of the nucleoside. For example,nucleosides comprising 2′-substituted or 2-modified sugar moieties arereferred to as 2′-substituted nucleosides or 2-modified nucleosides.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′bridging sugar substituents include but are not limited to: 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′ (“LNA”), 4′-CH₂—S-2′,4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referred to as “constrainedethyl” or “cEt” when in the S configuration), 4′-CH₂—O—CH₂-2′,4′-CH₂—N(R)-2′, 4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE” or “cMOE”) andanalogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhatet al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457,and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH₃)(CH₃)—O-2′ andanalogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283),4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., Prakash et al., U.S.Pat. No. 8,278,425), 4′-CH₂—O—N(CH₃)-2′ (see, e.g., Allerson et al.,U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745),4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74,118-134), 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see e.g., Seth et al.,U.S. Pat. No. 8,278,426), 4′-C(R_(a)R_(b))—N(R)—O-2′,4′-C(R_(a)R_(b))—O—N(R)-2′, 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′,wherein each R, R_(a), and R_(b) is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl (see, e.g. Imanishi et al., U.S. Pat. No.7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a))═C(R_(b))—,—C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—,—S(═O)_(x)—, and —N(R_(a))—;

-   -   wherein:    -   x is 0, 1, or 2;    -   n is 1, 2, 3, or 4;    -   each R_(a) and R_(b) is, independently, H, a protecting group,        hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂        alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted        C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,        heterocycle radical, substituted heterocycle radical,        heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,        substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,        N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl        (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is,        independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂        alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted        C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl        (C(═O)—H), substituted acyl, a heterocycle radical, a        substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted        C₁-C₁₂ aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, forexample: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443,Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97,5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., JAm. Chem. Soc., 2007, 129, 8362-8379; Elayadi et al., Curr. OpinionInvens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8,1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wengel etal., U.S. Pat. No. 7,053,207, Imanishi et al., U.S. Pat. No. 6,268,490,Imanishi et al. U.S. Pat. No. 6,770,748, Imanishi et al., U.S. RE44,779;Wengel et al., U.S. Pat. No. 6,794,499, Wengel et al., U.S. Pat. No.6,670,461; Wengel et al., U.S. Pat. No. 7,034,133, Wengel et al., U.S.Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel etal., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582;and Ramasamy et al., U.S. Pat. No. 6,525,191, Torsten et al., WO2004/106356, Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181;Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No.7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat.No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S.Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al.,U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth etal., U.S. Pat. No. 8,501,805; Allerson et al., US2008/0039618; andMigawa et al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, an LNA nucleoside (describedherein) may be in the α-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O-2′) or α-L-LNA bicyclic nucleosides have beenincorporated into oligonucleotides that showed antisense activity(Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein,general descriptions of bicyclic nucleosides include both isomericconfigurations. When the positions of specific bicyclic nucleosides(e.g., LNA or cEt) are identified in exemplified embodiments herein,they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the sugar moiety isreplaced, e.g., with a sulfur, carbon or nitrogen atom. In certain suchembodiments, such modified sugar moieties also comprise bridging and/ornon-bridging substituents as described herein. For example, certainsugar surrogates comprise a 4′-sulfur atom and a substitution at the2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat etal., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having otherthan 5 atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyransmay be further modified or substituted. Nucleosides comprising suchmodified tetrahydropyrans include but are not limited to hexitol nucleicacid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”)(see e.g., Leumann, C J. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA:

(“F-HNA”, see e.g., Swayze et al., U.S. Pat. No. 8,088,904; Swayze etal., U.S. Pat. No. 8,440,803; and Swayze et al., U.S. Pat. No.9,005,906, F-HNA can also be referred to as a F-THP or 3′-fluorotetrahydropyran), and nucleosides comprising additional modified THPcompounds having the formula:

wherein, independently, for each of said modified THP nucleoside:

-   -   Bx is a nucleobase moiety;    -   T₃ and T₄ are each, independently, an internucleoside linking        group linking the modified THP nucleoside to the remainder of an        oligonucleotide or one of T₃ and T₄ is an internucleoside        linking group linking the modified THP nucleoside to the        remainder of an oligonucleotide and the other of T₃ and T₄ is H,        a hydroxyl protecting group, a linked conjugate group, or a 5′        or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each,        independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆        alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or        substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is        independently selected from among: hydrogen, halogen,        substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,        OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁,        and each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided whereinq₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, atleast one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. Incertain embodiments, modified THP nucleosides are provided wherein oneof R₁ and R₂ is F. In certain embodiments, R₁ is F and R₂ is H, incertain embodiments, R₁ is methoxy and R₂ is H, and in certainembodiments, R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example, nucleosidescomprising morpholino sugar moieties and their use in oligonucleotideshave been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41,4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton etal., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444;and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term“morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties.Examples of nucleosides and oligonucleotides comprising such acyclicsugar surrogates include but are not limited to: peptide nucleic acid(“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org.Biomol. Chem., 2013, 11, 5853-5865), and nucleosides andoligonucleotides described in Manoharan et al., US2013/130378.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used in modified nucleosides.

2. Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications can impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.

In certain embodiments, compounds described herein comprise modifiedoligonucleotides. In certain embodiments, modified oligonucleotidescomprise one or more nucleoside comprising an unmodified nucleobase. Incertain embodiments, modified oligonucleotides comprise one or morenucleoside comprising a modified nucleobase. In certain embodiments,modified oligonucleotides comprise one or more nucleoside that does notcomprise a nucleobase, referred to as an abasic nucleoside.

In certain embodiments, modified nucleobases are selected from:5-substituted pyrimidines, 6-azapyrimi¬dines, alkyl or alkynylsubstituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6substituted purines. In certain embodiments, modified nucleobases areselected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine,5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine,6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-propynyl (C≡C—CH3) uracil,5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine,5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo,particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine,2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine,4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine,5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases. Furthermodified nucleobases include tricyclic pyrimidines, such as1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in Merigan et al., U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859;Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and thosedisclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T.,Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above notedmodified nucleobases as well as other modified nucleobases includewithout limitation, Manoharan et al., US2003/0158403, Manoharan et al.,US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al.,U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066;Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat.No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al.,U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cooket al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No.5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al.,U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No.5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S.Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook etal., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cooket al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903;Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No.5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al.,U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook etal., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No.6,005,096.

In certain embodiments, compounds targeted to a PCSK9 nucleic acidcomprise one or more modified nucleobases. In certain embodiments, themodified nucleobase is 5-methylcytosine. In certain embodiments, eachcytosine is a 5-methylcytosine.

3. Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage In certain embodiments, compounds describedherein having one or more modified, i.e. non-naturally occurring,internucleoside linkages are often selected over compounds havingnaturally occurring internucleoside linkages because of desirableproperties such as, for example, enhanced cellular uptake, enhancedaffinity for target nucleic acids, and increased stability in thepresence of nucleases.

In certain embodiments, compounds targeted to a PCSK9 nucleic acidcomprise one or more modified internucleoside linkages. In certainembodiments, the modified internucleoside linkages are phosphorothioatelinkages. In certain embodiments, each internucleoside linkage of anantisense compound is a phosphorothioate internucleoside linkage.

In certain embodiments, compounds described herein compriseoligonucleotides. Oligonucleotides having modified internucleosidelinkages include internucleoside linkages that retain a phosphorus atomas well as internucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, nucleosides of modified oligonucleotides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus-containinginternucleoside linkages include but are not limited to phosphates,which contain a phosphodiester bond (“P═O”) (also referred to asunmodified or naturally occurring linkages), phosphotriesters,methylphosphonates, phosphoramidates, and phosphorothioates (“P═S”), andphosphorodithioates (“HS—P═S”). Representative non-phosphorus containinginternucleoside linking groups include but are not limited tomethylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester, thionocarbamate(—O—C(═O)(NH)—S—); siloxane (—O—SiH2-O—); and N,N′-dimethylhydrazine(-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared tonaturally occurring phosphate linkages, can be used to alter, typicallyincrease, nuclease resistance of the oligonucleotide. In certainembodiments, internucleoside linkages having a chiral atom can beprepared as a racemic mixture, or as separate enantiomers.Representative chiral internucleoside linkages include but are notlimited to alkylphosphonates and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinginternucleoside linkages are well known to those skilled in the art.

Neutral internucleoside linkages include, without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH2-N(CH3)-O-5′), amide-3(3′-CH2-C(═O)—N(H)-5′), amide-4 (3′-CH2-N(H)—C(═O)-5′), formacetal(3′-O—CH2-O-5′), methoxypropyl, and thioformacetal (3′-S—CH2-O-5′).Further neutral internucleoside linkages include nonionic linkagescomprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide,sulfide, sulfonate ester and amides (See for example: CarbohydrateModifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds.,ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutralinternucleoside linkages include nonionic linkages comprising mixed N,O, S and CH2 component parts.

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif. In such embodiments, the internucleoside linkages in eachof two wing regions are different from the internucleoside linkages inthe gap region. In certain embodiments the internucleoside linkages inthe wings are phosphodiester and the internucleoside linkages in the gapare phosphorothioate. The nucleoside motif is independently selected, sosuch oligonucleotides having a gapped internucleoside linkage motif mayor may not have a gapped nucleoside motif and if it does have a gappednucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides comprise a region of uniformly modified internucleosidelinkages. In certain such embodiments, the oligonucleotide comprises aregion that is uniformly linked by phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide is uniformlylinked by phosphorothioate. In certain embodiments, each internucleosidelinkage of the oligonucleotide is selected from phosphodiester andphosphorothioate. In certain embodiments, each internucleoside linkageof the oligonucleotide is selected from phosphodiester andphosphorothioate and at least one internucleoside linkage isphosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

In certain embodiments, oligonucleotides comprise one or moremethylphosponate linkages. In certain embodiments, oligonucleotideshaving a gapmer nucleoside motif comprise a linkage motif comprising allphosphorothioate linkages except for one or two methylphosponatelinkages. In certain embodiments, one methylphosponate linkage is in thecentral gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number ofphosphorothioate internucleoside linkages and phosphodiesterinternucleoside linkages to maintain nuclease resistance. In certainembodiments, it is desirable to arrange the number and position ofphosphorothioate internucleoside linkages and the number and position ofphosphodiester internucleoside linkages to maintain nuclease resistance.In certain embodiments, the number of phosphorothioate internucleosidelinkages may be decreased and the number of phosphodiesterinternucleoside linkages may be increased. In certain embodiments, thenumber of phosphorothioate internucleoside linkages may be decreased andthe number of phosphodiester internucleoside linkages may be increasedwhile still maintaining nuclease resistance. In certain embodiments itis desirable to decrease the number of phosphorothioate internucleosidelinkages while retaining nuclease resistance. In certain embodiments itis desirable to increase the number of phosphodiester internucleosidelinkages while retaining nuclease resistance.

4. Certain Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. Oligonucleotides can have a motif, e.g. a pattern ofunmodified and/or modified sugar moieties, nucleobases, and/orinternucleoside linkages. In certain embodiments, modifiedoligonucleotides comprise one or more modified nucleoside comprising amodified sugar. In certain embodiments, modified oligonucleotidescomprise one or more modified nucleosides comprising a modifiednucleobase. In certain embodiments, modified oligonucleotides compriseone or more modified internucleoside linkage. In such embodiments, themodified, unmodified, and differently modified sugar moieties,nucleobases, and/or internucleoside linkages of a modifiedoligonucleotide define a pattern or motif. In certain embodiments, thepatterns of sugar moieties, nucleobases, and internucleoside linkagesare each independent of one another. Thus, a modified oligonucleotidemay be described by its sugar motif, nucleobase motif and/orinternucleoside linkage motif (as used herein, nucleobase motifdescribes the modifications to the nucleobases independent of thesequence of nucleobases).

a. Certain Sugar Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. In certain embodiments, oligonucleotides comprise oneor more type of modified sugar and/or unmodified sugar moiety arrangedalong the oligonucleotide or region thereof in a defined pattern orsugar motif. In certain instances, such sugar motifs include but are notlimited to any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides comprise or consist ofa region having a gapmer motif, which comprises two external regions or“wings” and a central or internal region or “gap.” The three regions ofa gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguoussequence of nucleosides wherein at least some of the sugar moieties ofthe nucleosides of each of the wings differ from at least some of thesugar moieties of the nucleosides of the gap. Specifically, at least thesugar moieties of the nucleosides of each wing that are closest to thegap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside ofthe 3′-wing) differ from the sugar moiety of the neighboring gapnucleosides, thus defining the boundary between the wings and the gap(i.e., the wing/gap junction). In certain embodiments, the sugarmoieties within the gap are the same as one another. In certainembodiments, the gap includes one or more nucleoside having a sugarmoiety that differs from the sugar moiety of one or more othernucleosides of the gap. In certain embodiments, the sugar motifs of thetwo wings are the same as one another (symmetric gapmer). In certainembodiments, the sugar motif of the 5′-wing differs from the sugar motifof the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides.In certain embodiments, the nucleosides of a gapmer are all modifiednucleosides.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides.In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 10 nucleosides. Incertain embodiment, each nucleoside of the gap of a gapmer is anunmodified 2′-deoxy nucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In suchembodiments, the nucleosides on the gap side of each wing/gap junctionare unmodified 2′-deoxy nucleosides and the nucleosides on the wingsides of each wing/gap junction are modified nucleosides. In certainsuch embodiments, each nucleoside of the gap is an unmodified 2′-deoxynucleoside. In certain such embodiments, each nucleoside of each wing isa modified nucleoside.

In certain embodiments, a modified oligonucleotide has a fully modifiedsugar motif wherein each nucleoside of the modified oligonucleotidecomprises a modified sugar moiety. In certain embodiments, modifiedoligonucleotides comprise or consist of a region having a fully modifiedsugar motif wherein each nucleoside of the region comprises a modifiedsugar moiety. In certain embodiments, modified oligonucleotides compriseor consist of a region having a fully modified sugar motif, wherein eachnucleoside within the fully modified region comprises the same modifiedsugar moiety, referred to herein as a uniformly modified sugar motif. Incertain embodiments, a fully modified oligonucleotide is a uniformlymodified oligonucleotide. In certain embodiments, each nucleoside of auniformly modified comprises the same 2′-modification.

In certain embodiments, a modified oligonucleotide can comprise a sugarmotif described in Swayze et al., US2010/0197762; Freier et al.,US2014/0107330; Freier et al., US2015/0184153; and Seth et al.,US2015/0267195, each of which is incorporated by reference in itsentirety herein.

b. Certain Nucleobase Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. In certain embodiments, oligonucleotides comprisemodified and/or unmodified nucleobases arranged along theoligonucleotide or region thereof in a defined pattern or motif. Incertain embodiments, each nucleobase is modified. In certainembodiments, none of the nucleobases are modified. In certainembodiments, each purine or each pyrimidine is modified. In certainembodiments, each adenine is modified. In certain embodiments, eachguanine is modified. In certain embodiments, each thymine is modified.In certain embodiments, each uracil is modified. In certain embodiments,each cytosine is modified. In certain embodiments, some or all of thecytosine nucleobases in a modified oligonucleotide are5-methylcytosines.

In certain embodiments, modified oligonucleotides comprise a block ofmodified nucleobases. In certain such embodiments, the block is at the3′-end of the oligonucleotide. In certain embodiments the block iswithin 3 nucleosides of the 3′-end of the oligonucleotide. In certainembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleosides of the 5′-end ofthe oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprisea nucleoside comprising a modified nucleobase. In certain suchembodiments, one nucleoside comprising a modified nucleobase is in thecentral gap of an oligonucleotide having a gapmer motif. In certain suchembodiments, the sugar moiety of said nucleoside is a 2′-deoxyribosylmoiety. In certain embodiments, the modified nucleobase is selectedfrom: a 2-thiopyrimidine and a 5-propynepyrimidine.

c. Certain Internucleoside Linkage Motifs

In certain embodiments, compounds described herein compriseoligonucleotides. In certain embodiments, oligonucleotides comprisemodified and/or unmodified internucleoside linkages arranged along theoligonucleotide or region thereof in a defined pattern or motif. Incertain embodiments, essentially each internucleoside linking group is aphosphate internucleoside linkage (P═O). In certain embodiments, eachinternucleoside linking group of a modified oligonucleotide is aphosphorothioate (P═S). In certain embodiments, each internucleosidelinking group of a modified oligonucleotide is independently selectedfrom a phosphorothioate and phosphate internucleoside linkage. Incertain embodiments, the sugar motif of a modified oligonucleotide is agapmer and the internucleoside linkages within the gap are all modified.In certain such embodiments, some or all of the internucleoside linkagesin the wings are unmodified phosphate linkages. In certain embodiments,the terminal internucleoside linkages are modified.

5. Certain Modified Oligonucleotides

In certain embodiments, compounds described herein comprise modifiedoligonucleotides. In certain embodiments, the above modifications(sugar, nucleobase, internucleoside linkage) are incorporated into amodified oligonucleotide. In certain embodiments, modifiedoligonucleotides are characterized by their modification, motifs, andoverall lengths. In certain embodiments, such parameters are eachindependent of one another. Thus, unless otherwise indicated, eachinternucleoside linkage of an oligonucleotide having a gapmer sugarmotif may be modified or unmodified and may or may not follow the gapmermodification pattern of the sugar modifications. For example, theinternucleoside linkages within the wing regions of a sugar gapmer maybe the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region of thesugar motif. Likewise, such gapmer oligonucleotides may comprise one ormore modified nucleobase independent of the gapmer pattern of the sugarmodifications. Furthermore, in certain instances, an oligonucleotide isdescribed by an overall length or range and by lengths or length rangesof two or more regions (e.g., a regions of nucleosides having specifiedsugar modifications), in such circumstances it may be possible to selectnumbers for each range that result in an oligonucleotide having anoverall length falling outside the specified range. In suchcircumstances, both elements must be satisfied. For example, in certainembodiments, a modified oligonucleotide consists of 15-20 linkednucleosides and has a sugar motif consisting of three regions, A, B, andC, wherein region A consists of 2-6 linked nucleosides having aspecified sugar motif, region B consists of 6-10 linked nucleosideshaving a specified sugar motif, and region C consists of 2-6 linkednucleosides having a specified sugar motif. Such embodiments do notinclude modified oligonucleotides where A and C each consist of 6 linkednucleosides and B consists of 10 linked nucleosides (even though thosenumbers of nucleosides are permitted within the requirements for A, B,and C) because the overall length of such oligonucleotide is 22, whichexceeds the upper limit of the overall length of the modifiedoligonucleotide (20). Herein, if a description of an oligonucleotide issilent with respect to one or more parameter, such parameter is notlimited. Thus, a modified oligonucleotide described only as having agapmer sugar motif without further description may have any length,internucleoside linkage motif, and nucleobase motif Unless otherwiseindicated, all modifications are independent of nucleobase sequence.

Certain Conjugated Compounds

In certain embodiments, the compounds described herein comprise orconsist of an oligonucleotide (modified or unmodified) and optionallyone or more conjugate groups and/or terminal groups. Conjugate groupsconsist of one or more conjugate moiety and a conjugate linker whichlinks the conjugate moiety to the oligonucleotide. Conjugate groups maybe attached to either or both ends of an oligonucleotide and/or at anyinternal position. In certain embodiments, conjugate groups are attachedto the 2′-position of a nucleoside of a modified oligonucleotide. Incertain embodiments, conjugate groups that are attached to either orboth ends of an oligonucleotide are terminal groups. In certain suchembodiments, conjugate groups or terminal groups are attached at the 3′and/or 5′-end of oligonucleotides. In certain such embodiments,conjugate groups (or terminal groups) are attached at the 3′-end ofoligonucleotides. In certain embodiments, conjugate groups are attachednear the 3′-end of oligonucleotides. In certain embodiments, conjugategroups (or terminal groups) are attached at the 5′-end ofoligonucleotides. In certain embodiments, conjugate groups are attachednear the 5′-end of oligonucleotides.

In certain embodiments, the oligonucleotide is modified. In certainembodiments, the oligonucleotide of a compound has a nucleobase sequencethat is complementary to a target nucleic acid. In certain embodiments,oligonucleotides are complementary to a messenger RNA (mRNA). In certainembodiments, oligonucleotides are complementary to a sense transcript.

Examples of terminal groups include but are not limited to conjugategroups, capping groups, phosphate moieties, protecting groups, modifiedor unmodified nucleosides, and two or more nucleosides that areindependently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to oneor more conjugate groups. In certain embodiments, conjugate groupsmodify one or more properties of the attached oligonucleotide, includingbut not limited to pharmacodynamics, pharmacokinetics, stability,binding, absorption, tissue distribution, cellular distribution,cellular uptake, charge and clearance In certain embodiments, conjugategroups impart a new property on the attached oligonucleotide, e.g.,fluorophores or reporter groups that enable detection of theoligonucleotide.

Certain conjugate groups and conjugate moieties have been describedpreviously, for example: cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3,2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanovet al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic, a palmityl moiety (Mishra et al.,Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, i, 923-937),_a tocopherol group (Nishina et al.,Molecular Therapy Nucleic Acids, 2015, 4, e220; doi:10.1038/mtna.2014.72 and Nishina et al., Molecular Therapy, 2008, 16,734-740), or a GalNAc cluster (e.g., WO2014/179620).

1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reportermolecules, polyamines, polyamides, peptides, carbohydrates (e.g.,GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers,cholesterols, thiocholesterols, cholic acid moieties, folate, lipids,phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores,and dyes.

In certain embodiments, a conjugate moiety comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugatelinkers. In certain compounds, a conjugate group is a single chemicalbond (i.e. conjugate moiety is attached to an oligonucleotide via aconjugate linker through a single bond). In certain embodiments, theconjugate linker comprises a chain structure, such as a hydrocarbylchain, or an oligomer of repeating units such as ethylene glycol,nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groupsselected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol,ether, thioether, and hydroxylamino. In certain such embodiments, theconjugate linker comprises groups selected from alkyl, amino, oxo, amideand ether groups. In certain embodiments, the conjugate linker comprisesgroups selected from alkyl and amide groups. In certain embodiments, theconjugate linker comprises groups selected from alkyl and ether groups.In certain embodiments, the conjugate linker comprises at least onephosphorus moiety. In certain embodiments, the conjugate linkercomprises at least one phosphate group. In certain embodiments, theconjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugatelinkers described above, are bifunctional linking moieties, e.g., thoseknown in the art to be useful for attaching conjugate groups to parentcompounds, such as the oligonucleotides provided herein. In general, abifunctional linking moiety comprises at least two functional groups.One of the functional groups is selected to bind to a particular site ona compound and the other is selected to bind to a conjugate group.Examples of functional groups used in a bifunctional linking moietyinclude but are not limited to electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In certain embodiments, bifunctional linking moieties compriseone or more groups selected from amino, hydroxyl, carboxylic acid,thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include but are not limited topyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include butare not limited to substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl or substituted orunsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferredsubstituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl.

In certain embodiments, conjugate linkers comprise 1-10linker-nucleosides. In certain embodiments, such linker-nucleosides aremodified nucleosides. In certain embodiments such linker-nucleosidescomprise a modified sugar moiety. In certain embodiments,linker-nucleosides are unmodified. In certain embodiments,linker-nucleosides comprise an optionally protected heterocyclic baseselected from a purine, substituted purine, pyrimidine or substitutedpyrimidine. In certain embodiments, a cleavable moiety is a nucleosideselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typicallydesirable for linker-nucleosides to be cleaved from the compound afterit reaches a target tissue. Accordingly, linker-nucleosides aretypically linked to one another and to the remainder of the compoundthrough cleavable bonds. In certain embodiments, such cleavable bondsare phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of theoligonucleotide. Accordingly, in embodiments in which a compoundcomprises an oligonucleotide consisting of a specified number or rangeof linked nucleosides and/or a specified percent complementarity to areference nucleic acid and the compound also comprises a conjugate groupcomprising a conjugate linker comprising linker-nucleosides, thoselinker-nucleosides are not counted toward the length of theoligonucleotide and are not used in determining the percentcomplementarity of the oligonucleotide for the reference nucleic acid.For example, a compound may comprise (1) a modified oligonucleotideconsisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10linker-nucleosides that are contiguous with the nucleosides of themodified oligonucleotide. The total number of contiguous linkednucleosides in such a compound is more than 30. Alternatively, ancompound may comprise a modified oligonucleotide consisting of 8-30nucleosides and no conjugate group. The total number of contiguouslinked nucleosides in such a compound is no more than 30. Unlessotherwise indicated conjugate linkers comprise no more than 10linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 5 linker-nucleosides. In certain embodiments, conjugatelinkers comprise no more than 3 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 2linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 1 linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to becleaved from the oligonucleotide. For example, in certain circumstancescompounds comprising a particular conjugate moiety are better taken upby a particular cell type, but once the compound has been taken up, itis desirable that the conjugate group be cleaved to release theunconjugated or parent oligonucleotide. Thus, certain conjugate maycomprise one or more cleavable moieties, typically within the conjugatelinker. In certain embodiments, a cleavable moiety is a cleavable bond.In certain embodiments, a cleavable moiety is a group of atomscomprising at least one cleavable bond. In certain embodiments, acleavable moiety comprises a group of atoms having one, two, three,four, or more than four cleavable bonds. In certain embodiments, acleavable moiety is selectively cleaved inside a cell or subcellularcompartment, such as a lysosome. In certain embodiments, a cleavablemoiety is selectively cleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: anamide, an ester, an ether, one or both esters of a phosphodiester, aphosphate ester, a carbamate, or a disulfide. In certain embodiments, acleavable bond is one or both of the esters of a phosphodiester. Incertain embodiments, a cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is aphosphate linkage between an oligonucleotide and a conjugate moiety orconjugate group.

In certain embodiments, a cleavable moiety comprises or consists of oneor more linker-nucleosides. In certain such embodiments, one or morelinker-nucleosides are linked to one another and/or to the remainder ofthe compound through cleavable bonds. In certain embodiments, suchcleavable bonds are unmodified phosphodiester bonds. In certainembodiments, a cleavable moiety is 2′-deoxy nucleoside that is attachedto either the 3′ or 5′-terminal nucleoside of an oligonucleotide by aphosphate internucleoside linkage and covalently attached to theremainder of the conjugate linker or conjugate moiety by a phosphate orphosphorothioate linkage. In certain such embodiments, the cleavablemoiety is 2′-deoxyadenosine.

3. Certain Cell-Targeting Conjugate Moieties

In certain embodiments, a conjugate group comprises a cell-targetingconjugate moiety. In certain embodiments, a conjugate group has thegeneral formula:

wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2or greater, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1 and k is 0. In certainembodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1,j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. Incertain embodiments, n is 2, j is 0 and k is 1. In certain embodiments,n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and kis 0. In certain embodiments, n is 3, j is 0 and k is 1. In certainembodiments, n is 3, j is 1 and k is 1.

In certain embodiments, conjugate groups comprise cell-targetingmoieties that have at least one tethered ligand. In certain embodiments,cell-targeting moieties comprise two tethered ligands covalentlyattached to a branching group. In certain embodiments, cell-targetingmoieties comprise three tethered ligands covalently attached to abranching group.

In certain embodiments, the cell-targeting moiety comprises a branchinggroup comprising one or more groups selected from alkyl, amino, oxo,amide, disulfide, polyethylene glycol, ether, thioether andhydroxylamino groups. In certain embodiments, the branching groupcomprises a branched aliphatic group comprising groups selected fromalkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether,thioether and hydroxylamino groups. In certain such embodiments, thebranched aliphatic group comprises groups selected from alkyl, amino,oxo, amide and ether groups. In certain such embodiments, the branchedaliphatic group comprises groups selected from alkyl, amino and ethergroups. In certain such embodiments, the branched aliphatic groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem.

In certain embodiments, each tether of a cell-targeting moiety comprisesone or more groups selected from alkyl, substituted alkyl, ether,thioether, disulfide, amino, oxo, amide, phosphodiester, andpolyethylene glycol, in any combination. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether, thioether, disulfide, amino, oxo, amide, andpolyethylene glycol, in any combination. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, phosphodiester, ether, amino, oxo, and amide, inany combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl,ether, amino, oxo, and amid, in any combination. In certain embodiments,each tether is a linear aliphatic group comprising one or more groupsselected from alkyl, amino, and oxo, in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and oxo, in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and phosphodiester, in any combination.In certain embodiments, each tether comprises at least one phosphoruslinking group or neutral linking group. In certain embodiments, eachtether comprises a chain from about 6 to about 20 atoms in length. Incertain embodiments, each tether comprises a chain from about 10 toabout 18 atoms in length. In certain embodiments, each tether comprisesabout 10 atoms in chain length.

In certain embodiments, each ligand of a cell-targeting moiety has anaffinity for at least one type of receptor on a target cell. In certainembodiments, each ligand has an affinity for at least one type ofreceptor on the surface of a mammalian liver cell. In certainembodiments, each ligand has an affinity for the hepaticasialoglycoprotein receptor (ASGP-R). In certain embodiments, eachligand is a carbohydrate. In certain embodiments, each ligand is,independently selected from galactose, N-acetyl galactoseamine (GalNAc),mannose, glucose, glucoseamine and fucose. In certain embodiments, eachligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, thecell-targeting moiety comprises 3 GalNAc ligands. In certainembodiments, the cell-targeting moiety comprises 2 GalNAc ligands. Incertain embodiments, the cell-targeting moiety comprises 1 GalNAcligand.

In certain embodiments, each ligand of a cell-targeting moiety is acarbohydrate, carbohydrate derivative, modified carbohydrate,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain such embodiments, the conjugate group comprises acarbohydrate cluster (see, e.g., Maier et al., “Synthesis of AntisenseOligonucleotides Conjugated to a Multivalent Carbohydrate Cluster forCellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29, or Rensenet al., “Design and Synthesis of Novel N-Acetylgalactosamine-TerminatedGlycolipids for Targeting of Lipoproteins to the HepaticAsiaglycoprotein Receptor,” J Med. Chem. 2004, 47, 5798-5808, which areincorporated herein by reference in their entirety). In certain suchembodiments, each ligand is an amino sugar or a thio sugar. For example,amino sugars may be selected from any number of compounds known in theart, such as sialic acid, α-D-galactosamine, β-muramic acid,2-deoxy-2-methylamino-L-glucopyranose,4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom 5-Thio-β-D-glucopyranose, methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, compounds described herein comprise a conjugategroup described herein as “LICA-1”. LICA-1 is shown below without theoptional cleavable moiety at the end of the conjugate linker:

In certain embodiments, compounds described herein comprise LICA-1 and acleavable moiety within the conjugate linker have the formula:

wherein oligo is an oligonucleotide.

Representative publications that teach the preparation of certain of theabove noted conjugate groups and compounds comprising conjugate groups,tethers, conjugate linkers, branching groups, ligands, cleavablemoieties as well as other modifications include without limitation, U.S.Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177,7,491,805, 8,106,022, 7,723,509, 9,127,276, US 2006/0148740, US2011/0123520, WO 2013/033230 and WO 2012/037254, Biessen et al., J. Med.Chem. 1995, 38, 1846-1852, Lee et al., Bioorganic & Medicinal Chemistry2011, 19, 2494-2500, Rensen et al., J. Biol. Chem. 2001, 276,37577-37584, Rensen et al., J. Med. Chem. 2004, 47, 5798-5808, Sliedregtet al., J. Med. Chem. 1999, 42, 609-618, and Valentijn et al.,Tetrahedron, 1997, 53, 759-770, each of which is incorporated byreference herein in its entirety.

In certain embodiments, compounds described herein comprise modifiedoligonucleotides comprising a gapmer or fully modified motif and aconjugate group comprising at least one, two, or three GalNAc ligands.In certain embodiments compounds described herein comprise a conjugategroup found in any of the following references: Lee, Carbohydr Res,1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945;Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al.,Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4,317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessenet al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron,1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490;Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol,2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584;Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al.,Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006,16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15,7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee etal., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; eachof which is incorporated by reference in its entirety.

Compositions and Methods for Formulating Pharmaceutical Compositions

Compounds described herein may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, or extent of disease.

Certain embodiments provide pharmaceutical compositions comprising oneor more compounds or a salt thereof. In certain embodiments, thecompounds are antisense compounds or oligomeric compounds. In certainembodiments, the compounds comprise or consist of a modifiedoligonucleotide. In certain such embodiments, the pharmaceuticalcomposition comprises a suitable pharmaceutically acceptable diluent orcarrier. In certain embodiments, a pharmaceutical composition comprisesa sterile saline solution and one or more compound. In certainembodiments, such pharmaceutical composition consists of a sterilesaline solution and one or more compound. In certain embodiments, thesterile saline is pharmaceutical grade saline. In certain embodiments, apharmaceutical composition comprises one or more compound and sterilewater. In certain embodiments, a pharmaceutical composition consists ofone compound and sterile water. In certain embodiments, the sterilewater is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more compound and sterilePBS. In certain embodiments, the sterile PBS is pharmaceutical gradePBS. Compositions and methods for the formulation of pharmaceuticalcompositions are dependent upon a number of criteria, including, but notlimited to, route of administration, or extent of disease.

A compound described herein targeted to PCSK9 nucleic acid can beutilized in pharmaceutical compositions by combining the compound with asuitable pharmaceutically acceptable diluent or carrier. In certainembodiments, a pharmaceutically acceptable diluent is water, such assterile water suitable for injection. Accordingly, in one embodiment,employed in the methods described herein is a pharmaceutical compositioncomprising a compound targeted to PCSK9 nucleic acid and apharmaceutically acceptable diluent. In certain embodiments, thepharmaceutically acceptable diluent is water. In certain embodiments,the compound comprises or consists of a modified oligonucleotideprovided herein.

Pharmaceutical compositions comprising compounds provided hereinencompass any pharmaceutically acceptable salts, esters, or salts ofsuch esters, or any other oligonucleotide which, upon administration toan animal, including a human, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof. Incertain embodiments, the compounds are antisense compounds or oligomericcompounds. In certain embodiments, the compound comprises or consists ofa modified oligonucleotide. Accordingly, for example, the disclosure isalso drawn to pharmaceutically acceptable salts of compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of a compound which are cleaved by endogenous nucleaseswithin the body, to form the active compound.

In certain embodiments, the compounds or compositions further comprise apharmaceutically acceptable carrier or diluent.

Certain Selected Compounds

Approximately 1540 newly designed compounds and a few previouslydisclosed compounds of various lengths, chemistries, and motifs weretested for their effect on human PCSK9 mRNA in vitro in several celltypes (Example 1-2). Of 1540 compounds tested for potency at a singledose in vitro, 183 selected compounds were tested for dose dependentinhibition in HepG2 cells (Example 2). Of the 183 compounds tested bydose response assays, 134 oligonucleotides were selected for single dosein vivo tolerability in rodents. These 134 oligonucleotides wereconjugated at the 3′-end with THA-C6-GalNAc3-(3R,5S)-5-(hydroxymethyl)pyrrolidin-3-ol phosphate endcap (henceforth referred to as 3′-THA).

In the single dose rodent tolerability model, BALB/c mice are dosed witha high concentration of 3′-THA oligonucleotide and liver functionmarkers, such as alanine transaminase and aspartate transaminase, aremeasured (Example 3). ISIS 863413 (SEQ ID NO: 1096), ISIS 863419 (SEQ IDNO: 1419), ISIS 863424 (SEQ ID NO: 1297), ISIS 863425 (SEQ ID NO: 1528),ISIS 863427 (SEQ ID NO: 1145), ISIS 863433 (SEQ ID NO: 1223), ISIS863434 (SEQ ID NO: 377), ISIS 863436 (SEQ ID NO: 763), ISIS 863437 (SEQID NO: 994), ISIS 863438 (SEQ ID NO: 1071), ISIS 863439 (SEQ ID NO:1147), ISIS 863441 (SEQ ID NO: 1302), ISIS 863444 (SEQ ID NO: 1149),ISIS 863445 (SEQ ID NO: 455), ISIS 863448 (SEQ ID NO: 1380), ISIS 863452(SEQ ID NO: 691), ISIS 863472 (SEQ ID NO: 926), ISIS 863473 (SEQ ID NO:1233), ISIS 863474 (SEQ ID NO: 619), ISIS 863475 (SEQ ID NO: 388), ISIS863477 (SEQ ID NO: 1542), ISIS 863479 (SEQ ID NO: 1158), ISIS 863480(SEQ ID NO: 1235), ISIS 863481 (SEQ ID NO: 389), ISIS 863482 (SEQ ID NO:1312), ISIS 863483 (SEQ ID NO: 1240), ISIS 863484 (SEQ ID NO: 549), ISIS863485 (SEQ ID NO: 550), ISIS 863486 (SEQ ID NO: 781), ISIS 863489 (SEQID NO: 939), ISIS 863490 (SEQ ID NO: 1016), ISIS 863491 (SEQ ID NO:1243), ISIS 863493 (SEQ ID NO: 629), ISIS 863494 (SEQ ID NO: 1017), ISIS863495 (SEQ ID NO: 1092), ISIS 863496 (SEQ ID NO: 1244), ISIS 863497(SEQ ID NO: 554), ISIS 863498 (SEQ ID NO: 478), ISIS 863499 (SEQ ID NO:1094), ISIS 863502 (SEQ ID NO: 563), ISIS 863506 (SEQ ID NO: 410), ISIS863507 (SEQ ID NO: 1411), ISIS 863509 (SEQ ID NO: 721), ISIS 863510 (SEQID NO: 1258), ISIS 863511 (SEQ ID NO: 645), ISIS 863512 (SEQ ID NO:955), ISIS 863514 (SEQ ID NO: 1413), ISIS 863516 (SEQ ID NO: 881), ISIS863517 (SEQ ID NO: 648), ISIS 863518 (SEQ ID NO: 725), ISIS 863520 (SEQID NO: 1111), ISIS 863522 (SEQ ID NO: 1188), ISIS 863524 (SEQ ID NO:885), ISIS 863525 (SEQ ID NO: 504), ISIS 863526 (SEQ ID NO: 1043), ISIS863527 (SEQ ID NO: 1195), ISIS 863531 (SEQ ID NO: 426), ISIS 863533 (SEQID NO: 427), ISIS 863536 (SEQ ID NO: 585), ISIS 863537 (SEQ ID NO:1047), ISIS 863538 (SEQ ID NO: 353), ISIS 863539 (SEQ ID NO: 1352), ISIS863541 (SEQ ID NO: 1203), ISIS 863545 (SEQ ID NO: 1516), ISIS 863547(SEQ ID NO: 1213), ISIS 863548 (SEQ ID NO: 367), ISIS 863549 (SEQ ID NO:1061), ISIS 863550 (SEQ ID NO: 523), ISIS 863552 (SEQ ID NO: 831), andISIS 863553 (SEQ ID NO: 908) were considered tolerable in this study andwere selected for further evaluation in a transgenic mouse model.

In the PCSK9 transgenic mice tolerability model, mice were dosed with3′-THA conjugated oligonucleotides and plasma levels of alaninetransaminase, aspartate transaminase, cholesterol, HDL-cholesterol,LDL-cholesterol and triglycerides were measured (Example 4). ISIS863433, ISIS 863490, ISIS 863512, ISIS 863527, ISIS 863538, ISIS 863425,ISIS 863438, ISIS 863439, ISIS 863444, ISIS 863413, ISIS 863434, ISIS863436, ISIS 863494, and ISIS 863539 were found tolerable. The selectedoligonucleotide sequences were conjugated at the 5′-end withTrishexylamino-(THA)-C6GalNAC3 endcap (henceforth referred to as 5′-THA)and tested in the CD-1 mouse model. The 5′THA conjugatedoligonucleotides are ISIS 845219, ISIS 863568, ISIS 863576, ISIS 863577,ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS863637, ISIS 863655, ISIS 863670, ISIS 863681, and ISIS 863682.

In the CD-1 mice tolerability model, mice were dosed for six weeks withvarying doses of antisense oligonucleotide and tolerability markers,such as liver and kidney function markers (plasma levels of ALT, AST,albumin, BUN, creatinine, and bilirubin), hematology markers (blood cellcounts for RBC, WBC, platelets, neutrophils, lymphocytes, and monocytes,as well as hemoglobin, hematocrit, and MCV content), and body and organweights were measured (Example 5). Only those oligonucleotides wereselected for further evaluation in the rat model, where treatmentyielded plasma levels of such markers which were within the expectedrange after treatment with antisense oligonucleotides. ISIS 863568, ISIS863576, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633,ISIS 863655, ISIS 863670, and ISIS 863681 were selected for furtherstudy in the rat model.

In the Sprague-Dawley rat model, similarly, the rats were dosed for 6weeks with antisense oligonucleotides and tolerability markers, such asliver function markers (plasma levels of ALT, AST, albumin, BUN,creatinine, and bilirubin), kidney function markers (urine levels ofcreatinine and total protein), and weekly body weights, and final organweights were measured (Example 6).

These oligonucleotides were also tested for their viscosity (Example 8)and all found to be optimal in their viscosity under the criteriatested. ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587,ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 were tested in adose-dependent four-week study in the PCSK9 transgenic mice model forefficacy (Example 7). At the end of 4 weeks, mice liver was analyzed forPCSK9 mRNA expression levels and the ED₅₀ was calculated. In addition,PCSK9 plasma protein levels, LDL-cholesterol plasma levels, andLDL-receptor levels in the liver were measured. It was observed thattreatment with the antisense oligonucleotides resulted in inhibition ofPCSK9 liver mRNA expression levels, reduction in LDL-cholesterol levelsin the plasma, and corresponding increase in LDL receptor levels in theliver. Specifically, ISIS 863633 treatment was found to be efficacious.

ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS863633, ISIS 863655, ISIS 863670, and ISIS 863681 were tested foractivity, pharmacokinetic profile and tolerability in a 12-week study incynomolgus monkeys (Example 9). Treatment with the compounds that werefully cross-reactive with the rhesus monkey gene sequence causedreduction of PCSK9 mRNA expression in liver tissue. Specifically,treatment with ISIS 863633, ISIS 863670, and ISIS 863681 caused areduction of PCSK9 mRNA expression in liver tissue, compared to the PBScontrol. It was noted that ISIS 863633 caused the highest reduction ofPCSK9 mRNA expression compared to the PBS control. Changes in PCSK9protein levels, LDL-cholesterol levels, total cholesterol levels, andhepatic LDL-receptor levels are a consequence of inhibition of PCSK9mRNA levels. Treatment with ISIS 863633, ISIS 863670, and ISIS 863681caused decreases in PCSK9 protein levels, LDL-cholesterol levels, totalcholesterol levels, and induction of hepatic LDL-receptor levels, withISIS 863633 causing the highest change. Treatment with the antisenseoligonucleotides did not cause any changes in HDL-cholesterol ortriglyceride levels. Hence, in terms of activity, ISIS 863633 was themost effective in the monkey study. Treatment with the compounds waswell tolerated in the monkeys, in particular, treatment with ISIS863633.

The new compounds were compared with previously designed compounds,including ISIS 405879 and ISIS 405995, which have previously beendetermined to be some of the most potent antisense compounds in vitro(see e.g., U.S. Pat. No. 8,084,437), as well as ISIS 431131 and ISIS480604, which have been previously described in U.S. Pat. No. 9,127,276(Example 10). The head-to-head comparison demonstrated that ISIS 863568,ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS863655, ISIS 863670, and ISIS 863681 were more efficacious than any ofthe previously publicly disclosed oligonucleotides.

Accordingly, provided herein are compounds with any one or more of theimproved properties. In certain embodiments, the compounds as describedherein are potent and tolerable.

EXAMPLES

The Examples below describe the screening process to identify leadcompounds targeted to PCSK9. Out of over 1540 oligonucleotides that werescreened, ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 emergedas the top lead compounds. In particular, ISIS 863633 exhibited the bestcombination of properties in terms of potency and tolerability out ofover 1540 oligonucleotides.

Non-Limiting Disclosure and Incorporation by Reference

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA). As provided herein, adesignation of ‘0’ for mRNA inhibition assays merely indicates that theantisense oligonucleotide did not inhibit mRNA expression levels.

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligonucleotide having the nucleobase sequence “ATCGATCG” encompassesany oligonucleotides having such nucleobase sequence, whether modifiedor unmodified, including, but not limited to, such compounds comprisingRNA bases, such as those having sequence “AUCGAUCG” and those havingsome DNA bases and some RNA bases such as “AUCGATCG” and compoundshaving other modified nucleobases, such as “AT^(m)CGAUCG,” wherein ^(m)Cindicates a cytosine base comprising a methyl group at the 5-position.

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Inhibition of Human PCSK9 in HepG2 Cells by 3-10-3cEt Gapmers

Antisense oligonucleotides were designed targeting a PCSK9 nucleic acidand were tested for their effects on PCSK9 mRNA in vitro. The chimericantisense oligonucleotides in the Tables below were designed as 3-10-3cEt gapmers. The gapmers are 16 nucleosides in length, wherein thecentral gap segment is comprised of ten 2′-deoxynucleosides and isflanked by wing segments on the 5′ end and the 3′ end of threenucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a cEt sugar modification. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each gapmer are5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer istargeted in the human gene sequence. “Stop site” indicates the 3′-mostnucleoside to which the gapmer is targeted human gene sequence. Eachgapmer listed in the Tables below is targeted to either the human PCSK9mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No.NM_174936.3) or the human PCSK9 genomic sequence, designated herein asSEQ ID NO: 2 (GENBANK Accession No. NC_000001.11 truncated fromnucleotides 55036001 to 55068000). ‘n/a’ indicates that the antisenseoligonucleotide does not target that particular gene sequence with 10000complementarity.

The antisense oligonucleotides were tested in a series of experimentsthat had similar culture conditions. The results for each experiment arepresented in separate tables shown below.

Study 1

Cultured HepG2 cells at a density of 20,000 cells per well weretransfected using electroporation with 1,000 nM antisenseoligonucleotide. ISIS 431131, previously disclosed in W 2014179620, wasalso included in the study as a benchmark oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and PCSK9 mRNA levels were measured by quantitative real-time PCR.Human PCSK9 primer probe set from ABI (ID4 Hs03037355_m1) was used tomeasure mRNA levels. PCSK9 mRNA levels were adjusted according to totalRNA content, as measured by RIBOGREEN®. Results are presented as percentinhibition of PCSK9, relative to untreated control cells. Several of thenewly designed oligonucleotides were more potent than the previouslydisclosed oligonucleotide, ISIS 431131.

TABLE 1 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmerstargeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: SEQISIS Start Stop % Start Stop ID No Site Site Sequence inhibition SiteSite NO 431131 1015 1034 GTCACACTTGCTGGCCTGTC 3 N/A N/A 3 859373 N/A N/ATCTGCCGTCCTTCCCA 36 4562 4577 4 859377 N/A N/A AGCATCTGCCGTCCTT 62 45664581 5 859381 N/A N/A TGTCAAGCACCACAGC 31 5010 5025 6 859385 N/A N/ATGACTTGTCAAGCACC 61 5015 5030 7 859389 N/A N/A ATAAGTGACTTGTCAA 45 50205035 8 859393 N/A N/A GCATTGTAAGTTCACT 84 5138 5153 9 859397 N/A N/AGCTTGCATTGTAAGTT 29 5142 5157 10 859401 N/A N/A GGTTTTCCCAGCTCTG 85 58975912 11 859405 N/A N/A CTCTGGTTTTCCCAGC 72 5901 5916 12 859409 N/A N/ACCACCTCTGGTTTTCC 67 5905 5920 13 859413 N/A N/A GTCTGCTCCAACTGCT 43 67146729 14 859417 N/A N/A CTCTTGTCTGCTCCAA 55 6719 6734 15 859421 N/A N/ATTTAGCTCTTGTCTGC 45 6724 6739 16 859425 N/A N/A GCAAGTCCCTGCTAGA 1811238 11253 17 859429 N/A N/A CACCTGCAAGTCCCTG 15 11243 11258 18 859433N/A N/A ATTGCCACCTGCAAGT 4 11248 11263 19 859437 N/A N/ATCATTCTCTCCTCAGG 8 11294 11309 20 859441 N/A N/A TGGACCCACCACATTG 3411383 11398 21 859445 N/A N/A TGCATGGACCCACCAC 61 11387 11402 22 859449N/A N/A CATGTTGCATGGACCC 61 11392 11407 23 859453 N/A N/AACAGTAGCCCCCCAAC 46 11608 11623 24 859457 N/A N/A TCACACAGTAGCCCCC 5211612 11627 25 859461 N/A N/A AAAGTCACACAGTAGC 51 11616 11631 26 859465N/A N/A GGTCACACAGTTTGGG 58 11639 11654 27 859469 N/A N/AACATATGCAAGGTCAC 74 11649 11664 28 859473 N/A N/A ACTCAGACATATGCAA 3211655 11670 29 859477 N/A N/A AGTGTCCCTCTTGGTC 59 11950 11965 30 859481N/A N/A TGCCAGTGTCCCTCTT 37 11954 11969 31 859485 N/A N/ATGATCCTCTGCCAGTG 58 11962 11977 32 859489 N/A N/A TCTGTGATCCTCTGCC 5011966 11981 33 859493 N/A N/A GGTCTCTGTGATCCTC 67 11970 11985 34 859497N/A N/A AGCTCTAGGGCCCATG 6 12671 12686 35 859501 N/A N/AAGCCAGCTCTAGGGCC 0 12675 12690 36 859505 N/A N/A GTGGGTGCTCAAAACG 2812924 12939 37 859509 N/A N/A AATAATGCCCCCGTAG 26 13176 13191 38 859513N/A N/A CTGGTTAATAATGCCC 47 13182 13197 39 859517 N/A N/ACTGGATACATTGGCAG 25 14125 14140 40 859521 N/A N/A CTCTGAGTGTGTTAGG 5414473 14488 41 859525 N/A N/A TGCTCTCTGAGTGTGT 26 14477 14492 42 859529N/A N/A GCATGCTGAGCAGGTC 78 14541 14556 43 859533 N/A N/ATGTAGCATGCTGAGCA 73 14545 14560 44 859537 N/A N/A ATTCAGTGGCTGCTCT 014755 14770 45 859541 N/A N/A GAGCAATTCAGTGGCT 24 14760 14775 46 859310 979  994 TCCTCGGGCACATTCT 34 16371 16386 47 468460 1075 1090TTGGCCACGCCGGCAT 8 16705 16720 48 859545 N/A N/A ATTTAGCAGCTACGGC 3517441 17456 49 859549 N/A N/A CAACTATTTAGCAGCT 36 17446 17461 50 859553N/A N/A AGGTTACCGCTGTCAG 73 20914 20929 51 859557 N/A N/AGACCTAGGTTACCGCT 33 20919 20934 52 859314 1498 1513 CCACTCTGTGACACAA 7921470 21485 53 859318 1503 1518 ATGTCCCACTCTGTGA 49 21475 21490 54859561 N/A N/A CTCGAAGGTAAGCCGC 66 21891 21906 55 859565 N/A N/ACAGCACTGTCCTGCAG 20 22880 22895 56 859569 N/A N/A TTTTTGCTGGTTTGAG 3423030 23045 57 859573 N/A N/A CACTTTTTTGCTGGTT 58 23034 23049 58 859577N/A N/A AATTCCACTTTTTTGC 15 23039 23054 59 859581 N/A N/AATCTGTGCACTATCCT 28 23069 23084 60 859585 N/A N/A TGGCATCTGTGCACTA 5523073 23088 61 859589 N/A N/A TCTCACACGAGCATTA 16 23176 23191 62 859593N/A N/A CTGCCTCTCACACGAG 46 23181 23196 63 859597 N/A N/ATCTTATGGAGGAGGAA 23 23853 23868 64 859601 N/A N/A TGTTTTCGACACAGGG 6824225 24240 65 859605 N/A N/A GGCATGTTTTCGACAC 68 24229 24244 66 859609N/A N/A CATCTTTTCAGTACTC 43 26516 26531 67 859613 N/A N/AATCGCATCTTTTCAGT 31 26520 26535 68 859322 2429 2444 CTGTCACTGGAGCTCC 2627572 27587 69 859325 2495 2510 GTCGGAACCATTTTAA 34 27638 27653 70859329 2501 2516 GGACAAGTCGGAACCA 29 27644 27659 71 859333 2505 2520AGAGGGACAAGTCGGA 61 27648 27663 72 859337 3461 3476 ATCTCCGCCAGGCCAG 2428604 28619 73 859341 3465 3480 AAGCATCTCCGCCAGG 68 28608 28623 74859345 3470 3485 CTTAGAAGCATCTCCG 61 28613 28628 75 859349 3476 3491CCATGCCTTAGAAGCA 58 28619 28634 76 859353 3538 3553 TGTCTGCTTGCTTGGG 6228681 28696 77 859357 3544 3559 GATAAATGTCTGCTTG 37 28687 28702 78859361 3593 3608 GTCTAGAAAAGTTGGC 63 28736 28751 79 859365 3597 3612ACAGGTCTAGAAAAGT 64 28740 28755 80 859369 3606 3621 AAAAGCAAAACAGGTC 3328749 28764 81

TABLE 2 SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start StopSEQ ISIS No Site Site Sequence inhibition Site Site ID NO 431131 10151034 GTCACACTTGCTGGCCTGTC 0 N/A N/A 3 859374 N/A N/A ATCTGCCGTCCTTCCC 424563 4578 82 859378 N/A N/A CAGCATCTGCCGTCCT 62 4567 4582 83 859382 N/AN/A CTTGTCAAGCACCACA 61 5012 5027 84 859386 N/A N/A GTGACTTGTCAAGCAC 105016 5031 85 859390 N/A N/A TGTAAGTTCACTCATG 56 5134 5149 86 859394 N/AN/A TGCATTGTAAGTTCAC 71 5139 5154 87 859398 N/A N/A GGGCTTGCATTGTAAG 135144 5159 88 859402 N/A N/A TGGTTTTCCCAGCTCT 78 5898 5913 89 859406 N/AN/A CCTCTGGTTTTCCCAG 61 5902 5917 90 859410 N/A N/A TCCACCTCTGGTTTTC 415906 5921 91 859414 N/A N/A TTGTCTGCTCCAACTG 39 6716 6731 92 859418 N/AN/A GCTCTTGTCTGCTCCA 71 6720 6735 93 859422 N/A N/A TATAAATCTCCCATCC 07280 7295 94 859426 N/A N/A CTGCAAGTCCCTGCTA 12 11240 11255 95 859430N/A N/A CCACCTGCAAGTCCCT 26 11244 11259 96 859434 N/A N/ACTCTCCTCAGGCCAGG 64 11289 11304 97 859438 N/A N/A CCCACCACATTGCATC 3711379 11394 98 859442 N/A N/A ATGGACCCACCACATT 10 11384 11399 99 859446N/A N/A TTGCATGGACCCACCA 40 11388 11403 100 859450 N/A N/ACGAGAACCGGTAAGGG 34 11447 11462 101 859454 N/A N/A CACAGTAGCCCCCCAA 3211609 11624 102 859458 N/A N/A GTCACACAGTAGCCCC 69 11613 11628 103859462 N/A N/A GCAAAGTCACACAGTA 64 11618 11633 104 859466 N/A N/AAGGTCACACAGTTTGG 83 11640 11655 105 859470 N/A N/A GACATATGCAAGGTCA 4011650 11665 106 859474 N/A N/A TCCCTCTTGGTCTCTG 55 11946 11961 107859478 N/A N/A CAGTGTCCCTCTTGGT 67 11951 11966 108 859482 N/A N/ACTGCCAGTGTCCCTCT 1 11955 11970 109 859486 N/A N/A GTGATCCTCTGCCAGT 3511963 11978 110 859490 N/A N/A CTCTGTGATCCTCTGC 68 11967 11982 111859494 N/A N/A GGGTCTCTGTGATCCT 33 11971 11986 112 859498 N/A N/ACAGCTCTAGGGCCCAT 56 12672 12687 113 859502 N/A N/A CAGCCAGCTCTAGGGC 3112676 12691 114 859506 N/A N/A AGTGGGTGCTCAAAAC 23 12925 12940 115859510 N/A N/A TTAATAATGCCCCCGT 32 13178 13193 116 859514 N/A N/ACCTGGTTAATAATGCC 36 13183 13198 117 859518 N/A N/A GAGTGTGTTAGGAGCT 8314469 14484 118 859522 N/A N/A TCTCTGAGTGTGTTAG 21 14474 14489 119859526 N/A N/A CTTGCTCTCTGAGTGT 42 14479 14494 120 859530 N/A N/AAGCATGCTGAGCAGGT 54 14542 14557 121 859534 N/A N/A CTGTAGCATGCTGAGC 7114546 14561 122 859538 N/A N/A AATTCAGTGGCTGCTC 27 14756 14771 123859542 N/A N/A AGAGCAATTCAGTGGC 42 14761 14776 124 859311  981  996CCTCCTCGGGCACATT 14 16373 16388 125 468460 1075 1090 TTGGCCACGCCGGCAT 016705 16720 48 859546 N/A N/A CTATTTAGCAGCTACG 57 17443 17458 126 859550N/A N/A TCAACTATTTAGCAGC 56 17447 17462 127 859554 N/A N/ATAGGTTACCGCTGTCA 47 20915 20930 128 859558 N/A N/A GGACCTAGGTTACCGC 4920920 20935 129 859315 1499 1514 CCCACTCTGTGACACA 94 21471 21486 130859319 1505 1520 TGATGTCCCACTCTGT 31 21477 21492 131 859562 N/A N/AGTCCTGCAGCCTCTAG 30 22873 22888 132 859566 N/A N/A CCAGCACTGTCCTGCA 5422881 22896 133 859570 N/A N/A TTTTTTGCTGGTTTGA 33 23031 23046 134859574 N/A N/A CCACTTTTTTGCTGGT 21 23035 23050 135 859578 N/A N/ACAATTCCACTTTTTTG 41 23040 23055 136 859582 N/A N/A CATCTGTGCACTATCC 4823070 23085 137 859586 N/A N/A ATGGCATCTGTGCACT 30 23074 23089 138859590 N/A N/A CCTCTCACACGAGCAT 48 23178 23193 139 859594 N/A N/ATCTGCCTCTCACACGA 29 23182 23197 140 859598 N/A N/A TTCGACACAGGGTAGC 6724221 24236 141 859602 N/A N/A ATGTTTTCGACACAGG 66 24226 24241 142859606 N/A N/A TGGGCATGTTTTCGAC 0 24231 24246 143 859610 N/A N/AGCATCTTTTCAGTACT 55 26517 26532 144 859614 N/A N/A CATCGCATCTTTTCAG 4026521 26536 145 468484 2430 2445 GCTGTCACTGGAGCTC 46 27573 27588 146859326 2497 2512 AAGTCGGAACCATTTT 56 27640 27655 147 859330 2502 2517GGGACAAGTCGGAACC 57 27645 27660 148 859334 3458 3473 TCCGCCAGGCCAGTGA 3328601 28616 149 859338 3462 3477 CATCTCCGCCAGGCCA 57 28605 28620 150859342 3466 3481 GAAGCATCTCCGCCAG 56 28609 28624 151 859346 3472 3487GCCTTAGAAGCATCTC 67 28615 28630 152 859350 3477 3492 ACCATGCCTTAGAAGC 4128620 28635 153 859354 3540 3555 AATGTCTGCTTGCTTG 69 28683 28698 154859358 3546 3561 AAGATAAATGTCTGCT 69 28689 28704 155 859362 3594 3609GGTCTAGAAAAGTTGG 51 28737 28752 156 859366 3602 3617 GCAAAACAGGTCTAGA 5228745 28760 157 859370 3632 3647 ACCCAGAATAAATATC 28 28775 28790 158

TABLE 3 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmerstargeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2:Start Stop % Start Stop SEQ ISIS No Site Site Sequence inhibition SiteSite ID NO 431131 1015 1034 GTCACACTTGCTGGCCTGTC 10 N/A N/A 3 859375 N/AN/A CATCTGCCGTCCTTCC 28 4564 4579 159 859379 N/A N/A CCAGCATCTGCCGTCC 554568 4583 160 859383 N/A N/A ACTTGTCAAGCACCAC 66 5013 5028 161 859387N/A N/A AGTGACTTGTCAAGCA 84 5017 5032 162 859391 N/A N/AATTGTAAGTTCACTCA 70 5136 5151 163 859395 N/A N/A TTGCATTGTAAGTTCA 825140 5155 164 859399 N/A N/A AAATTGAATGAATTGG 26 5768 5783 165 859403N/A N/A CTGGTTTTCCCAGCTC 73 5899 5914 166 859407 N/A N/AACCTCTGGTTTTCCCA 77 5903 5918 167 859411 N/A N/A TTCCACCTCTGGTTTT 235907 5922 168 859415 N/A N/A CTTGTCTGCTCCAACT 22 6717 6732 169 859419N/A N/A AGCTCTTGTCTGCTCC 81 6721 6736 170 859423 N/A N/AATTGAGGGAAAAAATC 12 7468 7483 171 859427 N/A N/A CCTGCAAGTCCCTGCT 4211241 11256 172 859431 N/A N/A GCCACCTGCAAGTCCC 47 11245 11260 173859435 N/A N/A ATTCTCTCCTCAGGCC 52 11292 11307 174 859439 N/A N/AGACCCACCACATTGCA 48 11381 11396 175 859443 N/A N/A CATGGACCCACCACAT 2511385 11400 176 859447 N/A N/A GTTGCATGGACCCACC 65 11389 11404 177859451 N/A N/A AGTAGCCCCCCAACTT 0 11606 11621 178 859455 N/A N/AACACAGTAGCCCCCCA 42 11610 11625 179 859459 N/A N/A AGTCACACAGTAGCCC 8311614 11629 180 859463 N/A N/A CACACAGTTTGGGTGT 37 11636 11651 181859467 N/A N/A ATATGCAAGGTCACAC 49 11647 11662 182 859471 N/A N/AAGACATATGCAAGGTC 46 11651 11666 183 859475 N/A N/A TGTCCCTCTTGGTCTC 6111948 11963 184 859479 N/A N/A CCAGTGTCCCTCTTGG 35 11952 11967 185859483 N/A N/A TCTGCCAGTGTCCCTC 27 11956 11971 186 859487 N/A N/ATGTGATCCTCTGCCAG 42 11964 11979 187 859491 N/A N/A TCTCTGTGATCCTCTG 7311968 11983 188 859495 N/A N/A TCTAGGGCCCATGCTT 21 12668 12683 189859499 N/A N/A CCAGCTCTAGGGCCCA 53 12673 12688 190 859503 N/A N/AGGCAGCCAGCTCTAGG 42 12678 12693 191 859507 N/A N/A CGCACAGTGGGTGCTC 4112930 12945 192 859511 N/A N/A GGTTAATAATGCCCCC 58 13180 13195 193859515 N/A N/A GCCTGGTTAATAATGC 0 13184 13199 194 859519 N/A N/ACTGAGTGTGTTAGGAG 52 14471 14486 195 859523 N/A N/A CTCTCTGAGTGTGTTA 5714475 14490 196 859527 N/A N/A TGCTGAGCAGGTCCTT 47 14538 14553 197859531 N/A N/A TAGCATGCTGAGCAGG 79 14543 14558 198 859535 N/A N/ATTCTGTAGCATGCTGA 50 14548 14563 199 859539 N/A N/A CAATTCAGTGGCTGCT 6214757 14772 200 859543 N/A N/A ACAGAGCAATTCAGTG 49 14763 14778 201468460 1075 1090 TTGGCCACGCCGGCAT 29 16705 16720 48 859547 N/A N/AACTATTTAGCAGCTAC 65 17444 17459 202 859551 N/A N/A GTCAACTATTTAGCAG 7317448 17463 203 859555 N/A N/A CTAGGTTACCGCTGTC 60 20916 20931 204859559 N/A N/A GGGACCTAGGTTACCG 31 20921 20936 205 859312 1495 1510CTCTGTGACACAAAGC 69 21467 21482 206 859316 1501 1516 GTCCCACTCTGTGACA 6621473 21488 207 859563 N/A N/A GCACTGTCCTGCAGCC 44 22878 22893 208859567 N/A N/A ATCCAGCACTGTCCTG 47 22883 22898 209 859571 N/A N/ACTTTTTTGCTGGTTTG 77 23032 23047 210 859575 N/A N/A TCCACTTTTTTGCTGG 023036 23051 211 859579 N/A N/A TGTGCACTATCCTGTA 39 23066 23081 212859583 N/A N/A GCATCTGTGCACTATC 73 23071 23086 213 859587 N/A N/AAGATGGCATCTGTGCA 65 23076 23091 214 859591 N/A N/A GCCTCTCACACGAGCA 5723179 23194 215 859595 N/A N/A ACTCTGCCTCTCACAC 27 23184 23199 216859599 N/A N/A TTTTCGACACAGGGTA 69 24223 24238 217 859603 N/A N/ACATGTTTTCGACACAG 69 24227 24242 218 859607 N/A N/A CTTTTCAGTACTCTAT 2826513 26528 219 859611 N/A N/A CGCATCTTTTCAGTAC 70 26518 26533 220859615 N/A N/A TCCATCGCATCTTTTC 51 26523 26538 221 859320 2425 2440CACTGGAGCTCCTGGG 45 27568 27583 222 859323 2431 2446 GGCTGTCACTGGAGCT 3327574 27589 223 859327 2498 2513 CAAGTCGGAACCATTT 52 27641 27656 224859331 2503 2518 AGGGACAAGTCGGAAC 28 27646 27661 225 859335 3459 3474CTCCGCCAGGCCAGTG 25 28602 28617 226 859339 3463 3478 GCATCTCCGCCAGGCC 5628606 28621 227 859343 3467 3482 AGAAGCATCTCCGCCA 63 28610 28625 228859347 3474 3489 ATGCCTTAGAAGCATC 8 28617 28632 229 859351 3478 3493GACCATGCCTTAGAAG 30 28621 28636 230 859355 3541 3556 AAATGTCTGCTTGCTT 5128684 28699 231 859359 3589 3604 AGAAAAGTTGGCTGTA 37 28732 28747 232859363 3595 3610 AGGTCTAGAAAAGTTG 74 28738 28753 233 859367 3604 3619AAGCAAAACAGGTCTA 37 28747 28762 234 859371 3633 3648 AACCCAGAATAAATAT 3028776 28791 235

TABLE 4 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmerstargeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2:Start Stop % Start Stop SEQ ISIS No Site Site Sequence inhibition SiteSite ID NO 431131 1015 1034 GTCACACTTGCTGGCCTGTC 0 N/A N/A 3 468460 10751090 TTGGCCACGCCGGCAT 25 16705 16720 48 859313 1497 1512CACTCTGTGACACAAA 59 21469 21484 236 859317 1502 1517 TGTCCCACTCTGTGAC 1021474 21489 237 859321 2428 2443 TGTCACTGGAGCTCCT 17 27571 27586 238859324 2432 2447 GGGCTGTCACTGGAGC 46 27575 27590 239 859328 2499 2514ACAAGTCGGAACCATT 6 27642 27657 240 859332 2504 2519 GAGGGACAAGTCGGAA 127647 27662 241 859336 3460 3475 TCTCCGCCAGGCCAGT 20 28603 28618 242859340 3464 3479 AGCATCTCCGCCAGGC 60 28607 28622 243 859344 3468 3483TAGAAGCATCTCCGCC 59 28611 28626 244 859348 3475 3490 CATGCCTTAGAAGCAT 028618 28633 245 859352 3480 3495 CCGACCATGCCTTAGA 63 28623 28638 246859356 3542 3557 TAAATGTCTGCTTGCT 67 28685 28700 247 859360 3591 3606CTAGAAAAGTTGGCTG 59 28734 28749 248 859364 3596 3611 CAGGTCTAGAAAAGTT 4428739 28754 249 859368 3605 3620 AAAGCAAAACAGGTCT 62 28748 28763 250859372 N/A N/A TGCCGTCCTTCCCACC 17 4560 4575 251 859376 N/A N/AGCATCTGCCGTCCTTC 40 4565 4580 252 859380 N/A N/A TCCCAGCATCTGCCGT 494570 4585 253 859384 N/A N/A GACTTGTCAAGCACCA 83 5014 5029 254 859388N/A N/A AAGTGACTTGTCAAGC 77 5018 5033 255 859392 N/A N/ACATTGTAAGTTCACTC 77 5137 5152 256 859396 N/A N/A CTTGCATTGTAAGTTC 655141 5156 257 859400 N/A N/A TTTCCCAGCTCTGTAT 28 5894 5909 258 859404N/A N/A TCTGGTTTTCCCAGCT 48 5900 5915 259 859408 N/A N/ACACCTCTGGTTTTCCC 78 5904 5919 260 859412 N/A N/A CTTCCACCTCTGGTTT 285908 5923 261 859416 N/A N/A TCTTGTCTGCTCCAAC 13 6718 6733 262 859420N/A N/A TAGCTCTTGTCTGCTC 39 6722 6737 263 859424 N/A N/AACCCTAGGTGTACTTT 27 7779 7794 264 859428 N/A N/A ACCTGCAAGTCCCTGC 3111242 11257 265 859432 N/A N/A TGCCACCTGCAAGTCC 44 11246 11261 266859436 N/A N/A CATTCTCTCCTCAGGC 38 11293 11308 267 859440 N/A N/AGGACCCACCACATTGC 19 11382 11397 268 859444 N/A N/A GCATGGACCCACCACA 5611386 11401 269 859448 N/A N/A TGTTGCATGGACCCAC 53 11390 11405 270859452 N/A N/A CAGTAGCCCCCCAACT 19 11607 11622 271 859456 N/A N/ACACACAGTAGCCCCCC 56 11611 11626 272 859460 N/A N/A AAGTCACACAGTAGCC 3211615 11630 273 859464 N/A N/A GTCACACAGTTTGGGT 69 11638 11653 274859468 N/A N/A CATATGCAAGGTCACA 59 11648 11663 275 859472 N/A N/ATCAGACATATGCAAGG 72 11653 11668 276 859476 N/A N/A GTGTCCCTCTTGGTCT 6711949 11964 277 859480 N/A N/A GCCAGTGTCCCTCTTG 46 11953 11968 278859484 N/A N/A CCTCTGCCAGTGTCCC 34 11958 11973 279 859488 N/A N/ACTGTGATCCTCTGCCA 58 11965 11980 280 859492 N/A N/A GTCTCTGTGATCCTCT 7111969 11984 281 859496 N/A N/A GCTCTAGGGCCCATGC 16 12670 12685 282859500 N/A N/A GCCAGCTCTAGGGCCC 16 12674 12689 283 859504 N/A N/AAGAGAAATGCATGCTA 68 12841 12856 284 859508 N/A N/A GGCGCACAGTGGGTGC 012932 12947 285 859512 N/A N/A TGGTTAATAATGCCCC 40 13181 13196 286859516 N/A N/A TTGCCTGGTTAATAAT 12 13186 13201 287 859520 N/A N/ATCTGAGTGTGTTAGGA 62 14472 14487 288 859524 N/A N/A GCTCTCTGAGTGTGTT 5614476 14491 289 859528 N/A N/A CATGCTGAGCAGGTCC 66 14540 14555 290859532 N/A N/A GTAGCATGCTGAGCAG 79 14544 14559 291 859536 N/A N/ATCAGTGGCTGCTCTGA 24 14753 14768 292 859540 N/A N/A GCAATTCAGTGGCTGC 3314758 14773 293 859544 N/A N/A TAGTTAACACACAGAA 17 16208 16223 294859548 N/A N/A AACTATTTAGCAGCTA 43 17445 17460 295 859552 N/A N/ACGTCAACTATTTAGCA 66 17449 17464 296 859556 N/A N/A CCTAGGTTACCGCTGT 3320917 20932 297 859560 N/A N/A GCACAGACCCTGACTG 23 21567 21582 298859564 N/A N/A AGCACTGTCCTGCAGC 48 22879 22894 299 859568 N/A N/ATTTTGCTGGTTTGAGA 52 23029 23044 300 859572 N/A N/A ACTTTTTTGCTGGTTT 7023033 23048 301 859576 N/A N/A ATTCCACTTTTTTGCT 24 23038 23053 302859580 N/A N/A TCTGTGCACTATCCTG 38 23068 23083 303 859584 N/A N/AGGCATCTGTGCACTAT 75 23072 23087 304 859588 N/A N/A TCACACGAGCATTAAG 3423174 23189 305 859592 N/A N/A TGCCTCTCACACGAGC 38 23180 23195 306859596 N/A N/A TAGACAGGATCAACTC 18 23406 23421 307 859600 N/A N/AGTTTTCGACACAGGGT 61 24224 24239 308 859604 N/A N/A GCATGTTTTCGACACA 6324228 24243 309 859608 N/A N/A ATCTTTTCAGTACTCT 48 26515 26530 310859612 N/A N/A TCGCATCTTTTCAGTA 31 26519 26534 311 859616 N/A N/AAGCAGCTAGGCCACAG 20 26841 26856 312

Study 2

Cultured HepG2 cells at a density of 20,000 cells per well weretransfected using electroporation with 3,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and PCSK9 mRNA levels were measured byquantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID4Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of PCSK9, relative tountreated control cells.

TABLE 5 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmerstargeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2:Start Stop % Start Stop SEQ ISIS No Site Site Sequence inhibition SiteSite ID NO 848304  757  772 AACTTCAAGGCCAGCT 66 N/A N/A 313 848215  108 123 CTGAACTGAACGGCGG 68 3583 3598 314 848231  320  335 GGCGAGGAGACCTAGA87 3795 3810 315 848247  485  500 ACGCAAGGCTAGCACC 77 3960 3975 316848788 N/A N/A GGCAAGCCCGCTTTCT 88 4456 4471 317 848804 N/A N/ACCCTTTTAAAGATTAG 67 5053 5068 318 848820 N/A N/A CCAGAGAAGAAACATC 625524 5539 319 848836 N/A N/A AAGAATCTGGAGCTGC 84 6430 6445 320 848852N/A N/A CACTGAGGACCAAATT 65 6806 6821 321 848868 N/A N/AGCTATATAAAGGAATA 65 7161 7176 322 848884 N/A N/A CATGAGCAAGTCACTC 657387 7402 323 848900 N/A N/A AGTATAGTAGATGATA 82 7720 7735 324 848262614 629 CGAGAGGTGGGTCTCC 54 7887 7902 325 466848 673 688GTGAGGTATCCCCGGC 88 7946 7961 326 848289 689 704 GACATGCAGGATCTTG 797962 7977 327 848916 N/A N/A GGGAATTCTATACAGA 72 8193 8208 328 848932N/A N/A CACCGAAGATGTGACA 51 8521 8536 329 848948 N/A N/AAAGGGAAAGGCCTGAG 53 9040 9055 330 848964 N/A N/A CCCCACAGGCAGCCTC 619610 9625 331 848980 N/A N/A CCGGAGGACAGACTAG 27 10368 10383 332 848319 862  877 TGGTATTCATCCGCCC 60 10623 10638 333 848996 N/A N/AAGCCAAACGGAGCTGG 51 11093 11108 334 849012 N/A N/A AAAGGAACAGGCTCTT 5611842 11857 335 849028 N/A N/A CTCTAGGGCCCATGCT 56 12669 12684 336849044 N/A N/A GGATACACAGGCTCGC 96 13108 13123 337 849060 N/A N/AGATGATGTGACCACTG 86 13864 13879 338 849076 N/A N/A GGCCAGCAAGGTGGGC 3514277 14292 339 849092 N/A N/A TGCTAGTAGGTCTGGG 72 14675 14690 340849108 N/A N/A CCAAAGGAAGACTTCA 70 15400 15415 341 849124 N/A N/AATTTAAAGACTCAAAG 16 15870 15885 342 849140 N/A N/A ACATTTGTGGGAGAGG 5616250 16265 343 848334  984  999 CGTCCTCCTCGGGCAC 85 16376 16391 344848348 1018 1033 TCACACTTGCTGGCCT 52 16648 16663 345 848364 1061 1076ATCCCGGCCGCTGACC 57 16691 16706 346 468460 1075 1090 TTGGCCACGCCGGCAT 4816705 16720 48 848379 1085 1100 GCTGGCACCCTTGGCC 57 16715 16730 347849156 N/A N/A AAGACATAAAGACATC 89 17083 17098 348 849172 N/A N/ACAGAAGGTTATTGATT 68 17735 17750 349 849188 N/A N/A TGCTAGTTATTAAGCA 1518080 18095 350 849204 N/A N/A CAGTAACAGCTCTTTT 80 19020 19035 351849220 N/A N/A CCTTATTATCCCTTTC 70 19396 19411 352 849236 N/A N/ACAACATCAAATTCTGC 92 19658 19673 353 848395 1165 1180 TTCCGAATAAACTCCA 7319996 20011 354 848411 1345 1360 ACCTCGGGAGCTGAGG 20 20176 20191 355849252 N/A N/A GATGGAGGTTTCGAGC 87 20381 20396 356 849268 N/A N/ATATCAAGTGGTTCTAA 44 21039 21054 357 848427 1458 1473 CACCAATGATGTCCTC 4521430 21445 358 849284 N/A N/A TCTTATCGGCCAGGTG 75 21810 21825 359848443 1608 1623 TGGCAGAGAAGTGGAT 21 22101 22116 360 848459 1730 1745CCATACAGTCCTGCAA 40 22512 22527 361 848475 1807 1822 GAGCAGCTCAGCAGCT 4422589 22604 362 849300 N/A N/A TACAGAAGAGCTGGAG 61 22767 22782 363849316 N/A N/A ACACAGAGTGGTTTCA 79 23262 23277 364 848489 1951 1966CTGCAGTTGGCCTGGG 57 23571 23586 365 848505 2006 2021 GTGGCAGTGGACACGG 7123626 23641 366 849332 N/A N/A AGGAGAAGTAAGGTCA 91 23828 23843 367849348 N/A N/A GGGCATGTTTTCGACA 65 24230 24245 368 849364 N/A N/ATTCCACCCAGAGATGG 34 25105 25120 369 849380 N/A N/A CGGTATGGTGGTGGCA 7125630 25645 370 849396 N/A N/A GGGCAAGTGGATCCAA 38 26364 26379 371849412 N/A N/A CCACACTTCATTTCTC 35 27298 27313 372 848521 2225 2240GCAGGCCACGGTCACC 67 27368 27383 373 848537 2483 2498 TTAAAGCTCAGCCCCA 6727626 27641 374 848553 2560 2575 CCCCGGAAAGGCGGAA 39 27703 27718 375848569 2704 2719 CCGAGCACAGCTCGAC 62 27847 27862 376 848584 2739 2754ACGGACATCGGCACAT 92 27882 27897 377 848600 2776 2791 GGCACGGAACAAGAGC 8727919 27934 378 848616 2888 2903 ACCTTTCACACTCACC 92 28031 28046 379468491 2971 2986 TGCCATCCAGAAAGCT 64 28114 28129 380 848646 3085 3100CACCTTTGGGTGTTGC 80 28228 28243 381 848662 3132 3147 CCACTGCACACTGCCG 8728275 28290 382 848678 3220 3235 TGGTTCCAGGTTTCTT 87 28363 28378 383848693 3253 3268 CCTGCTGTGTGAGCTT 93 28396 28411 384 848709 3290 3305CTTCAGAGCCAGCCCA 65 28433 28448 385 848725 3380 3395 CCGAGCTTCCTGGTCT 9228523 28538 386 848741 3433 3448 GGGTGATAACGGAAAA 62 28576 28591 387848756 3547 3562 AAAGATAAATGTCTGC 92 28690 28705 388 848772 3631 3646CCCAGAATAAATATCT 89 28774 28789 389

TABLE 6Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop PCSK9 Start Stop SEQISIS No Site Site Sequence (% inhibition) Site Site ID NO 848216  123 138 CTCCAGGCTCAGACCC 82  3598  3613 390 848232  353  368GCCCATGAGGGCCAGG 67  3828  3843 391 848248  486  501 AACGCAAGGCTAGCAC 62 3961  3976 392 848789 N/A N/A CTATAATGGCAAGCCC 76  4463  4478 393848805 N/A N/A CGTAGGGACGATTGTC 65  5073  5088 394 848821 N/A N/AATGAACTTGAGGAGGC 85  5558  5573 395 848837 N/A N/A TTGGAAGAATCTGGAG 81 6434  6449 396 848853 N/A N/A CAACACTGAGGACCAA 77  6809  6824 397848869 N/A N/A GGCAACACTTCTTAAA 60  7219  7234 398 848885 N/A N/AGTGCAGCCATGAGCAA 73  7394  7409 399 848901 N/A N/A ACAGAGTATAGTAGAT 73 7724  7739 400 848263  616  631 TGCGAGAGGTGGGTCT 42  7889  7904 401848275  674  689 GGTGAGGTATCCCCGG 67  7947  7962 402 848290  690  705AGACATGCAGGATCTT 56  7963  7978 403 848917 N/A N/A TCAAATGAAGATAGAC 68 8237  8252 404 848933 N/A N/A GAATACCCAGTCCCCT 49  8548  8563 405848949 N/A N/A CTCAAGGGAAAGGCCT 59  9043  9058 406 848965 N/A N/ACATGGCAGCGGTGAAC 47  9638  9653 407 848981 N/A N/A GATCAAACCTGTCCCC 4210427 10442 408 848997 N/A N/A TGCAGACCGTTTTCCA 83 11221 11236 409849013 N/A N/A GGAAAGGAACAGGCTC 88 11844 11859 410 849029 N/A N/ATTTGAGGGCGGCAGCC 32 12687 12702 411 849045 N/A N/A TGCTGGATACACAGGC 7913112 13127 412 849061 N/A N/A TGTACCTGGGTTCTGC 86 13885 13900 413849077 N/A N/A CACTAGATATTGAGCT 55 14329 14344 414 849093 N/A N/ACTATGCTAGTAGGTCT 51 14678 14693 415 849109 N/A N/A GGCCAAAGGAAGACTT 2215402 15417 416 849125 N/A N/A CTGCAAGAAAGACAAC 73 1588 15904 417 848320 887  902 CACCAGGCTGCCTCCG 49 16279 16294 418 848335  985 1000CCGTCCTCCTCGGGCA  8 16377 16392 419 849141 N/A N/A CATCAGACGGCCGTGC 4016416 16431 420 848349 1019 1034 GTCACACTTGCTGGCC 62 16649 16664 421468459 1063 1078 GCATCCCGGCCGCTGA 76 16693 16708 422 468460 1075 1090TTGGCCACGCCGGCAT 46 16705 16720  48 848380 1091 1106 GCGCATGCTGGCACCC 4516721 16736 423 849157 N/A N/A GATAACATAACAAAAG 29 17096 17111 424849173 N/A N/A GCAGAAGGTTATTGAT 64 17736 17751 425 849189 N/A N/AACAGCTGCTAGTTATT 96 18085 18100 426 18398 18413 849205 N/A N/ATGCTACTGTCAACAGT 86 19032 19047 427 849221 N/A N/A CAATAACCTTATTATC  619402 19417 428 849237 N/A N/A AGAGAACAGACTGAGG 83 19727 19742 429848396 1170 1185 GGCTTTTCCGAATAAA 43 20001 20016 430 849253 N/A N/AGGCAGAGGACGCAGGG 40 20538 20553 431 849269 N/A N/A ACTATTCGGTGTATCA 8221050 21065 432 848428 1466 1481 GCTGGAGGCACCAATG  0 21438 21453 433849285 N/A N/A GTCTTATCGGCCAGGT 86 21811 21826 434 848444 1617 1632TGACATCTTTGGCAGA 39 22110 22125 435 848460 1731 1746 ACCATACAGTCCTGCA 1922513 22528 436 848476 1814 1829 GAAACTGGAGCAGCTC 54 22596 22611 437849301 N/A N/A CATTGAAAATCCATCC 83 22895 22910 438 849317 N/A N/ACCCAAGGAAGACTGTT 56 23318 23333 439 848490 1977 1992 CCTCAGCTGGTGGAGC 5423597 23612 440 848506 2007 2022 GGTGGCAGTGGACACG 46 23627 23642 441849333 N/A N/A AGGTAATACCTTTTTC 51 23871 23886 442 849349 N/A N/ACACCATCCCTTGATGC 46 24285 24300 443 849365 N/A N/A GAAAACACCATCTTTC 2625118 25133 444 849381 N/A N/A GGCGGTATGGTGGTGG 71 25632 25647 445849397 N/A N/A ACCCAGGGCAAGTGGA 56 26369 26384 446 849413 N/A N/ACCCCACCCACACTTCA 53 27304 27319 447 848522 2227 2242 TCGCAGGCCACGGTCA 6927370 27385 448 848538 2485 2500 TTTTAAAGCTCAGCCC 38 27628 27643 449848554 2565 2580 AGCAGCCCCGGAAAGG 67 27708 27723 450 848570 2706 2721ACCCGAGCACAGCTCG 90 27849 27864 451 848585 2741 2756 CCACGGACATCGGCAC 9327884 27899 452 848601 2777 2792 TGGCACGGAACAAGAG 54 27920 27935 453848617 2898 2913 GGCCATCAGCACCTTT 63 28041 28056 454 848632 2976 2991CTAGATGCCATCCAGA 91 28119 28134 455 848647 3088 3103 GGCCACCTTTGGGTGT 4628231 28246 456 848663 3141 3156 GTGCATGCACCACTGC 78 28284 28299 457848679 3221 3236 CTGGTTCCAGGTTTCT 88 28364 28379 458 848694 3254 3269TCCTGCTGTGTGAGCT 91 28397 28412 459 848710 3302 3317 GAAGAGGCTTGGCTTC 1728445 28460 460 848726 3381 3396 ACCGAGCTTCCTGGTC 38 28524 28539 461848742 3438 3453 GGCCTGGGTGATAACG 26 28581 28596 462 848757 3550 3565CCAAAAGATAAATGTC 46 28693 28708 463 848773 3638 3653 TACAAAACCCAGAATA 5928781 28796 464 848305  758  773 CAACTTCAAGGCCAGC 79 N/A N/A 465 8484121349 1364 GATGACCTCGGGAGCT 38 N/A N/A 466

Study 3

Cultured HepG2 cells at a density of 20,000 cells per well weretransfected using electroporation with 1,000 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and PCSK9 mRNA levels were measured byquantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID4Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of PCSK9, relative tountreated control cells.

TABLE 7Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop PCSK9 Start Stop SEQISIS No Site Site Sequence (% inhibition) Site Site ID NO 848205   4  19 CACCAGAGCCCCATCG  3  3479  3494 467 848221  189  204CTGGGAGCCGCTGCTG 42  3664  3679 468 848237  389  404 TGGCAGCGGCCACCAG  0 3864  3879 469 848253  506  521 GGCCAGGCCGTCCTCC 51  3981  3996 470848778 N/A N/A CCGCACCTTGGCGCAG  0  4035  4050 471 848794 N/A N/AGAAACAGATGGAATAC 28  4626  4641 472 848810 N/A N/A ACCCAGCACACTCAGA  0  529  5306 473 848826 N/A N/A GTACTCTGTGCAGTGG 80  5748  5763 474848842 N/A N/A GAGTAGAACAGAGTCC 54  6544  6559 475 848858 N/A N/ATTAATAATCAGCCTTC 46  6933  6948 476 848874 N/A N/A TACAAATGCAGGCAGA 40 7256  7271 477 848890 N/A N/A CTCGACAACAGGTTTT 65  7577  7592 478848906 N/A N/A AGGAACATGATGACAT  0  7811  7826 479 848268  644  659CTGCAGGCGGCGGGCA  0  7917  7932 480 848279  679  694 ATCTTGGTGAGGTATC 40 7952  7967 481 848294  695  710 ATGGAAGACATGCAGG 44  7968  7983 482848922 N/A N/A TAAAATGACTCAGGCT 19  8406  8421 483 848938 N/A N/ACATCAAGTTAGAGGCC 49  8650  8665 484 848954 N/A N/A TATACGGGTACCTTCT 28 9143  9158 485 848970 N/A N/A GCTCAGTGCAAACTGC  0 10094 10109 486848309  779  794 CTCGATGTAGTCGACA 62 10540 10555 487 848986 N/A N/ACCCGAGAAGTGGAAAC 12 10729 10744 488 849002 N/A N/A CCAAGATCCCACGAGA 5611458 11473 489 849018 N/A N/A GAGAAAGTGGTCCTGC 35 12122 12137 490849034 N/A N/A CAGCAATAACTGATTT 42 12825 12840 491 849044 N/A N/AGGATACACAGGCTCGC 83 13108 13123 337 849050 N/A N/A CGCCTGAGAAGCTCAG 4413353 13368 492 849066 N/A N/A CCACACAACGCACATC 15 14043 14058 493849082 N/A N/A GACCAAACAGTGCTCG 28 14360 14375 494 849098 N/A N/ACTACAAAGACCTTTTC  0 14849 14864 495 849114 N/A N/A TAGGAGAAAGTAGGGA 2615533 15548 496 849130 N/A N/A GAATATCAATATCTAA 19 15983 15998 497848325  931  946 TCCCGGTGGTCACTCT  2 16323 16338 498 848340  990 1005GGGTCCCGTCCTCCTC 38 16382 16397 499 849146 N/A N/A CCACATGAGAAAGACC  016614 16629 500 848354 1026 1041 CATGACTGTCACACTT 23 16656 16671 501848369 1074 1089 TGGCCACGCCGGCATC 17 16704 16719 502 468460 1075 1090TTGGCCACGCCGGCAT  4 16705 16720  48 848385 1114 1129 TGGCAGTTGAGCACGC  016744 16759 503 849162 N/A N/A GTCTAGAAAAAGTCCT 65 17257 17272 504849178 N/A N/A CCGTGCCAGGTCATGC 78 17957 17972 505 849194 N/A N/ACTGGGATACAGACACC 42 18254 18269 506 849210 N/A N/A AATTAAAAGACTCCAT 1819154 19169 507 849226 N/A N/A TATTAGCAATTACACC 45 19502 19517 508849242 N/A N/A TTACAGGCACAGAGTG 25 19914 19929 509 848401 1228 1243CTGTACCCACCCGCCA 11 20059 20074 510 849258 N/A N/A AACAAGATTCCTTCCC 3020814 20829 511 849274 N/A N/A GGCAGAACTCTGGCAC 56 21163 21178 512848417 1402 1417 CCCAAAGTCCCCAGGG  0 21374 21389 513 849290 N/A N/AGGTGAAAGATGGTGAT 10 22003 22018 514 848433 1544 1559 CATCATGGCTGCAATG  022037 22052 515 848449 1636 1651 GGGAACCAGGCCTCAT 25 22129 22144 516848465 1736 1751 TGCTGACCATACAGTC  0 22518 22533 517 849306 N/A N/AGTGCACTATCCTGTAG 28 23065 23080 518 849322 N/A N/A CAAAGACGGAAATGGG 4423462 23477 519 848481 1925 1940 GCACCTGGCAATGGCG 34 23545 23560 520848495 1993 2008 CGGGTCCCCATGCTGG  0 23613 23628 521 849338 N/A N/ATGCTTAGCACTCATCA  0 24102 24117 522 849354 N/A N/A CCTACATGCCAGCCTG 7324472 24487 523 849370 N/A N/A AAAGAGATGCTGGCCT 28 25228 25243 524848511 2082 2097 GCACAGGCGGCTTGTG 28 25413 25428 525 849386 N/A N/ACCATATTTATGCACAT 62 25806 25821 526 849402 N/A N/A TTTCAGTACTCTATAT 1226511 26526 527 848527 2372 2387 AACGGCTGTCACGGCC 42 27515 27530 528848543 2507 2522 AGAGAGGGACAAGTCG 47 27650 27665 529 848559 2603 2618TTCCAGGCAAGGAGGC 23 27746 27761 530 848575 2712 2727 GGCAGCACCCGAGCAC 7227855 27870 531 848590 2757 2772 ATAAAAGTCATTCTGC 15 27900 27915 532848606 2787 2802 ATTGAATGCCTGGCAC 36 27930 27945 533 848622 2919 2934CCACAGTTAGCTGGAG 45 28062 28077 534 848637 3036 3051 GCTCAGGAAACCAAGG 1228179 28194 535 848652 3122 3137 CTGCCGAGTCAGTCCT 66 28265 28280 536848668 3172 3187 TGCCGGGTAGTGGAGC 49 28315 28330 537 848683 3226 3241CCCCTCTGGTTCCAGG 43 28369 28384 538 848699 3261 3276 GCTCAGTTCCTGCTGT 1828404 28419 539 848715 3317 3332 AGCCGGGTGAAGTAAG 26 28460 28475 540848731 3386 3401 CACTCACCGAGCTTCC 73 28529 28544 541 848747 3471 3486CCTTAGAAGCATCTCC 52 28614 28629 542 848762 3576 3591 GTAAAAAGGCAACAGA 3628719 28734 543

TABLE 8Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 848206    7  22CGCCACCAGAGCCCCA 30  3482  3497 544 848222  207  222 CGGAATCCTGGCTGGG 55 3682  3697 545 848238  476  491 TAGCACCAGCTCCTCG 53  3951  3966 546848254  520  535 TGCTCGGGTGCTTCGG 35  3995  4010 547 848779 N/A N/AGGCTGCGGGTTCGCCC 13  4070  4085 548 848795 N/A N/A CGAGAATACCTCCGCC 73 4682  4697 549 848811 N/A N/A GCTGAGTAAGGACTTG 64  5317  5332 550848827 N/A N/A TTCCGCTAAATAAAAA  9  5781  5796 551 848843 N/A N/ATCAGAGTAGAACAGAG 51  6547  6562 552 848859 N/A N/A GAGGGAGGTGCCAAGC 31 6954  6969 553 848875 N/A N/A CTACAAATGCAGGCAG 63  7257  7272 554848891 N/A N/A AACTACGGGCCACACT  7  7591  7606 555 848907 N/A N/AACGGATCCTGGCCCCA 45  7834  7849 556 848269  663  678 CCCGGCGGGCAGCCTG 18 7936  7951 557 848280  680  695 GATCTTGGTGAGGTAT  1  7953  7968 558848295  699  714 GGCCATGGAAGACATG 18  7972  7987 559 848923 N/A N/ACTAGAGTCATGCTTTT  0  8425  8440 560 848939 N/A N/A GTGGAGAATCAGTGTG 40 8683  8698 561 848955 N/A N/A TTGAAGTCCAGCTCTC 16  9258  9273 562848971 N/A N/A GCAATTCGGTTTGTCC 65 10140 10155 563 848310  795  810AGACAGAGGAGTCCTC 31 10556 10571 564 848987 N/A N/A GATCATTTAAGGCAAG 2610850 10865 565 849003 N/A N/A TCCCAAGATCCCACGA  5 11460 11475 566849019 N/A N/A CAAGAGAAGCTTCTCC 20 12152 12167 567 849035 N/A N/ACTACAGCAATAACTGA 46 12828 12843 568 849044 N/A N/A GGATACACAGGCTCGC 7813108 13123 337 849051 N/A N/A GAGCAGGGAGCTCATT  0 13395 13410 569849067 N/A N/A CTAGAAGACAGCACAG 15 14085 14100 570 849083 N/A N/ACCGACCTGAAGACATC 41 14380 14395 571 849099 N/A N/A GCCCACCGCATAATCC 1814897 14912 572 849115 N/A N/A GACAAAGGTTAGGGTA 46 15549 15564 573849131 N/A N/A TGAATATCAATATCTA 30 15984 15999 574 848326  939  954CCTCGATTTCCCGGTG 30 16331 16346 575 848341  992 1007 GCGGGTCCCGTCCTCC  016384 16399 576 849147 N/A N/A GACCACATGAGAAAGA  0 16616 16631 577848355 1029 1044 TGCCATGACTGTCACA 35 16659 16674 578 468460 1075 1090TTGGCCACGCCGGCAT 19 16705 16720  48 848370 1076 1091 CTTGGCCACGCCGGCA  016706 16721 579 848386 1133 1148 GCTAACCGTGCCCTTC 20 16763 16778 580849163 N/A N/A CAGTAAGGGAGAGAAC  3 17363 17378 581 849179 N/A N/ACCCGTGCCAGGTCATG 38 17958 17973 582 849195 N/A N/A AGCTGCTAGTTATTTA 4418396 18411 583 849211 N/A N/A GAAACAGGGACAGTTG 25 19189 19204 584849227 N/A N/A GGAAGATATTAGCAAT 78 19508 19523 585 849243 N/A N/ACTTACAGGCACAGAGT 27 19915 19930 586 848402 1238 1253 GAGGACGCGGCTGTAC 2520069 20084 587 849259 N/A N/A GTGGAACAAGATTCCT 40 20818 20833 588849275 N/A N/A AGGCAGAACTCTGGCA 56 21164 21179 589 848418 1414 1429CCAAAGTTGGTCCCCA  0 21386 21401 590 848434 1546 1561 AGCATCATGGCTGCAA 4922039 22054 591 848450 1641 1656 CCTCAGGGAACCAGGC 28 22134 22149 592849291 N/A N/A TGCCATCCTGCTTACC 30 22209 22224 593 848466 1738 1753TGTGCTGACCATACAG 20 22520 22535 594 849307 N/A N/A CACCAAAATGCTGCAA 3923094 23109 595 849323 N/A N/A GGCCTTAGAGTCAAAG  0 23473 23488 596468479 1926 1941 AGCACCTGGCAATGGC 47 23546 23561 597 848496 1997 2012GACACGGGTCCCCATG 28 23617 23632 598 849339 N/A N/A ACTAAGCTAAGTGCTT  724113 24128 599 849355 N/A N/A GCAAAATGGTGCTCTT 52 24690 24705 600849371 N/A N/A ACCTAGAAACAACTCA 23 25251 25266 601 848512 2084 2099CAGCACAGGCGGCTTG 14 25415 25430 602 849387 N/A N/A ATGCATATTGCATTTC 4425828 25843 603 849403 N/A N/A GGCCAGACCACACTCC 13 26808 26823 604848528 2375 2390 GGCAACGGCTGTCACG 52 27518 27533 605 848544 2509 2524TGAGAGAGGGACAAGT 27 27652 27667 606 848560 2613 2628 AGTGAGTGAGTTCCAG 6527756 27771 607 848576 2713 2728 TGGCAGCACCCGAGCA 56 27856 27871 608848591 2761 2776 CTCAATAAAAGTCATT 45 27904 27919 609 848607 2791 2806GAGGATTGAATGCCTG 37 27934 27949 610 848623 2921 2936 CTCCACAGTTAGCTGG 3528064 28079 611 848638 3045 3060 GTAAAGGTGGCTCAGG 46 28188 28203 612848653 3123 3138 ACTGCCGAGTCAGTCC 29 28266 28281 613 848669 3180 3195GTGTACCCTGCCGGGT  3 28323 28338 614 848684 3227 3242 CCCCCTCTGGTTCCAG 5428370 28385 615 848700 3263 3278 TGGCTCAGTTCCTGCT 36 28406 28421 616848716 3321 3336 GCCCAGCCGGGTGAAG 26 28464 28479 617 848732 3388 3403ATCACTCACCGAGCTT 69 28531 28546 618 848748 3473 3488 TGCCTTAGAAGCATCT 6828616 28631 619 848763 3578 3593 CTGTAAAAAGGCAACA 52 28721 28736 620

TABLE 9Inhibition of PCSK9 mRNA by 3-10-3 gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 848207   12   27GATCACGCCACCAGAG  0  3487  3502 621 848223  209  224 CGCGGAATCCTGGCTG  1 3684  3699 622 848239  477  492 CTAGCACCAGCTCCTC 38  3952  3967 623848255  533  548 GGCTGTGGTTCCGTGC 29  4008  4023 624 848780 N/A N/AAAACAGCACCGCACCG  0  4090  4105 625 848796 N/A N/A CCGCACACGGTCGGCA 35 4821  4836 626 848812 N/A N/A GAACAAGGAAGAGGGC 39  5341  5356 627848828 N/A N/A CAAAGACCCATCTGAA 32  5803  5818 628 848844 N/A N/ATCATGAATCAAGTCCA 71  6650  6665 629 848860 N/A N/A GCAGAGATCAATCACA 36 6997  7012 630 848876 N/A N/A TCCCATCCAGATGCTC 34  7272  7287 631848892 N/A N/A ATTCAGGCAAAAATGG 12  7608  7623 632 848270  665  680TCCCCGGCGGGCAGCC 24  7938  7953 633 848281  681  696 GGATCTTGGTGAGGTA 23 7954  7969 634 848296  704  719 AAGAAGGCCATGGAAG 38  7977  7992 635848908 N/A N/A TGCCATTCCCAAAAAG  0  8045  8060 636 848924 N/A N/ACAAAGGAATTTTGGAC  0  8442  8457 637 848940 N/A N/A TGCCATGTGGAGAATC 24 8689  8704 638 848956 N/A N/A TCGAACCCAGGCTGGT  0  9286  9301 639848972 N/A N/A CATGACATAACAGCAC 24 10158 10173 640 848311  797  812AAAGACAGAGGAGTCC 28 10558 10573 641 848988 N/A N/A AAAGAGGATCATTTAA  010856 10871 642 849004 N/A N/A CAAGAACGACAAAGCT 48 11493 11508 643849020 N/A N/A GGACACGGAAGAGGCA 63 12166 12181 644 849036 N/A N/AACAGAGAAATGCATGC 66 12843 12858 645 849044 N/A N/A GGATACACAGGCTCGC  813108 13123 337 849052 N/A N/A CTCTATCTTCCAAACC 22 13578 13593 646849068 N/A N/A ATACAGGCTTATCTAG  3 14097 14112 647 849084 N/A N/ACAATAGGCATCTACCA 59 14444 14459 648 849100 N/A N/A AGGACTTACAGCCCAC 1214907 14922 649 849116 N/A N/A GCCCATGCAGGACAAA 11 15559 15574 650849132 N/A N/A TATGAATATCAATATC  0 15986 16001 651 468453  950  965CATGACCCTGCCCTCG 45 16342 16357 652 848342  995 1010 GAAGCGGGTCCCGTCC 1716387 16402 653 849148 N/A N/A ACACAAGGACCACATG  0 16623 16638 654848356 1040 1055 TGCCAGGTGGGTGCCA 26 16670 16685 655 468460 1075 1090TTGGCCACGCCGGCAT  0 16705 16720  48 848371 1077 1092 CCTTGGCCACGCCGGC 3716707 16722 656 848387 1135 1150 CCGCTAACCGTGCCCT 17 16765 16780 657849164 N/A N/A CTTGAGCTGTGCGACC 27 17497 17512 658 849180 N/A N/ACCCCGTGCCAGGTCAT 26 17959 17974 659 849196 N/A N/A ACAACAGCCACATTTA 5618413 18428 660 849212 N/A N/A TGTGAAACAGGGACAG 17 19192 19207 661849228 N/A N/A TAATATATACATCCTA  0 19564 19579 662 849244 N/A N/ACCTTACAGGCACAGAG 16 19916 19931 663 848403 1241 1256 GTTGAGGACGCGGCTG 1920072 20087 664 849260 N/A N/A CCAACGCGCGCGCGCG  4 20870 20885 665849276 N/A N/A GAAAATCTGACTGCCC 43 21180 21195 666 848419 1416 1431GGCCAAAGTTGGTCCC  0 21388 21403 667 848435 1550 1565 AGACAGCATCATGGCT 4322043 22058 668 848451 1656 1671 TCAGTACCCGCTGGTC 41 22149 22164 669849292 N/A N/A GGTCACACAGACCTCC 24 22252 22267 670 848467 1744 1759CCCGAGTGTGCTGACC 39 22526 22541 671 849308 N/A N/A TTCCACCAAAATGCTG  723097 23112 672 848482 1927 1942 CAGCACCTGGCAATGG 10 23547 23562 673848497 1998 2013 GGACACGGGTCCCCAT 32 23618 23633 674 849324 N/A N/AGGCAAGCCCAGCCTCC  0 23667 23682 675 849340 N/A N/A AACGATAGCTAGAATT 1224143 24158 676 849356 N/A N/A TGGTAGGGTTGTGGTT  0 24782 24797 677849372 N/A N/A GCAAGAGCTAGGAAAC 60 25265 25280 678 848513 2086 2101CTCAGCACAGGCGGCT 41 25417 25432 679 849388 N/A N/A ATGCATGCATATTGCA  025832 25847 680 849404 N/A N/A CTTCAGACTCCAGCCT  0 26944 26959 681848529 2378 2393 GATGGCAACGGCTGTC  0 27521 27536 682 848545 2511 2526GCTGAGAGAGGGACAA 54 27654 27669 683 848561 2617 2632 CCAGAGTGAGTGAGTT 3027760 27775 684 848577 2714 2729 CTGGCAGCACCCGAGC 52 27857 27872 685848592 2762 2777 GCTCAATAAAAGTCAT 44 27905 27920 686 848608 2795 2810ACCTGAGGATTGAATG 16 27938 27953 687 848624 2944 2959 AATCAGGGAGCCCCCA 028087 28102 688 848639 3047 3062 GAGTAAAGGTGGCTCA 19 28190 28205 689848654 3124 3139 CACTGCCGAGTCAGTC 51 28267 28282 690 848670 3186 3201GCGAATGTGTACCCTG 81 28329 28344 691 848685 3229 3244 CGCCCCCTCTGGTTCC 5728372 28387 692 848701 3264 3279 CTGGCTCAGTTCCTGC 69 28407 28422 693848717 3330 3345 AAATGAGGAGCCCAGC 42 28473 28488 694 848733 3392 3407TGCCATCACTCACCGA 48 28535 28550 695 848749 3482 3497 CCCCGACCATGCCTTA 4328625 28640 696 848764 3580 3595 GGCTGTAAAAAGGCAA 68 28723 28738 697

TABLE 10Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 848208   15   30GCAGATCACGCCACCA  0  3490  3505 698 848224  212  227 GCGCGCGGAATCCTGG 21 3687  3702 699 848240  478  493 GCTAGCACCAGCTCCT 37  3953  3968 700468441  541  556 TGGAAGGTGGCTGTGG  0  4016  4031 701 848781 N/A N/ACCGAGAGGAAACAGCA 52  4098  4113 702 848797 N/A N/A AGCAAACTCGCCCCGC 55 4833  4848 703 848813 N/A N/A GGCTCAGGGAACAAGG 39  5349  5364 704848829 N/A N/A CACCATCATCGCTGAA 39  5834  5849 705 848845 N/A N/AGCAGACATACCTGCTT 24  6693  6708 706 848861 N/A N/A CTACATGTGCAGAGAT 52 7005  7020 707 848877 N/A N/A ATAAATCTCCCATCCA 36  7279  7294 708848893 N/A N/A TTTCAAATGTGCCATT 22  7621  7636 709 468443  666  681ATCCCCGGCGGGCAGC 52  7939  7954 710 848282  682  697 AGGATCTTGGTGAGGT 47 7955  7970 711 848297  732  747 CGCCACTCATCTTCAC 23  8005  8020 712848909 N/A N/A GGCCTACTAAGCACAG 49  8100  8115 713 848925 N/A N/ATTCAAAGGAATTTTGG  0  8444  8459 714 848941 N/A N/A ACCCAGGTCCAGACTC 44 8793  8808 715 848957 N/A N/A GGTTAGACAGCCAATA 48  9318  9333 716848973 N/A N/A CTAAATCATGACATAA 14 10164 10179 717 848312  799  814GCAAAGACAGAGGAGT 46 10560 10575 718 848989 N/A N/A TCCAAGGTAAGTGCAG 5210924 10939 719 849005 N/A N/A GACAAGAACGACAAAG 46 11495 11510 720849021 N/A N/A GACAATGAAGAGGAGA 69 12240 12255 721 849037 N/A N/ACTCAATACCTGACAGA 50 12854 12869 722 849044 N/A N/A GGATACACAGGCTCGC 813108 13123 337 849053 N/A N/A TGGTAAAAGCCCCCAC 39 13677 13692 723849069 N/A N/A ATACATTGGCAGACAG 56 14121 14136 724 849085 N/A N/AACTCATCAATAGGCAT 79 14450 14465 725 849101 N/A N/A CATCACAGGACTTACA  014913 14928 726 849117 N/A N/A CAAGAGACTCACTGGG 16 15581 15596 727849133 N/A N/A TGCCAAGAAGGACCCA 61 16020 16035 728 848327  953  968GACCATGACCCTGCCC 41 16345 16360 729 848343  997 1012 TGGAAGCGGGTCCCGT 1116389 16404 730 849149 N/A N/A TGCTCGACGAACACAA  0 16633 16648 731848357 1053 1068 CGCTGACCACCCCTGC 33 16683 16698 732 468460 1075 1090TTGGCCACGCCGGCAT  7 16705 16720  48 848372 1078 1093 CCCTTGGCCACGCCGG 4916708 16723 733 848388 1137 1152 TGCCGCTAACCGTGCC 10 16767 16782 734849165 N/A N/A GTGGAAGTCAAGCTGC 42 17550 17565 735 849181 N/A N/AGCCCCGTGCCAGGTCA 58 17960 17975 736 849197 N/A N/A TAGGAGACAGCTAGTG 2518486 18501 737 849213 N/A N/A CACAAACTTGAACAGA 38 19248 19263 738849229 N/A N/A CTTAATATATACATCC  0 19566 19581 739 849245 N/A N/AGCCAAGGTCACCCCTT  0 19963 19978 740 848404 1283 1298 GACCAGCACGACCCCA 1320114 20129 741 849261 N/A N/A ACCTAGGTTACCGCTG 33 20918 20933 742849277 N/A N/A AGGAAAATCTGACTGC 26 21182 21197 743 848420 1423 1438ACACAGCGGCCAAAGT  0 21395 21410 744 848436 1552 1567 GCAGACAGCATCATGG 7622045 22060 745 848452 1658 1673 GGTCAGTACCCGCTGG 29 22151 22166 746849293 N/A N/A CCTATTTAAGGTGGCG 5 22311 22326 747 848468 1753 1768CGTGTAGGCCCCGAGT 44 22535 22550 748 849309 N/A N/A CAGAAATGCCTGCCCC 6423141 23156 749 848483 1928 1943 GCAGCACCTGGCAATG  0 23548 23563 750848498 1999 2014 TGGACACGGGTCCCCA  2 23619 23634 751 849325 N/A N/ACCTCACCCCAGGGCAA  0 23678 23693 752 849341 N/A N/A GTAACGATAGCTAGAA 5224145 24160 753 849357 N/A N/A TGAACATGGTAGGGTT 62 24788 24803 754849373 N/A N/A AGGCAAGAGCTAGGAA 51 25267 25282 755 848514 2113 2128ACGCACTGGTTGGGCT  5 25444 25459 756 849389 N/A N/A GTTAAATAGATCAGAG 1525989 26004 757 849405 N/A N/A CCCCATAGCCTGCCCC  0 27058 27073 758848530 2381 2396 GCAGATGGCAACGGCT 14 27524 27539 759 848546 2520 2535CCATGGAGGGCTGAGA  3 27663 27678 760 848562 2619 2634 ACCCAGAGTGAGTGAG 2927762 27777 761 848578 2715 2730 GCTGGCAGCACCCGAG 18 27858 27873 762848593 2763 2778 AGCTCAATAAAAGTCA 61 27906 27921 763 848609 2820 2835GAAGAATCCTGCCTCC 42 27963 27978 764 848625 2946 2961 TTAATCAGGGAGCCCC 3128089 28104 765 848640 3050 3065 GCAGAGTAAAGGTGGC 38 28193 28208 766848655 3125 3140 ACACTGCCGAGTCAGT 32 28268 28283 767 848671 3188 3203GTGCGAATGTGTACCC 68 28331 28346 768 848686 3237 3252 GGCAGGCACGCCCCCT 1728380 28395 769 848702 3265 3280 TCTGGCTCAGTTCCTG 51 28408 28423 770848718 3332 3347 AAAAATGAGGAGCCCA 17 28475 28490 771 848734 3415 3430TTCCATGCCTGCAGGC 35 28558 28573 772 848750 3502 3517 GGACAGTTGTTGGCCC 2528645 28660 773 848765 3588 3603 GAAAAGTTGGCTGTAA 22 28731 28746 774

TABLE 11Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 848209   51   66ACCTTCTAGGGTGTGG  0  3526  3541 775 848225  242  257 CTGAAGTTCAGGAGCA 35 3717  3732 776 848241  479  494 GGCTAGCACCAGCTCC 58  3954  3969 777848256  549  564 CGCAGCGGTGGAAGGT 15  4024  4039 778 848782 N/A N/AGCGAAGAGCCCTCGGC 33  4158  4173 779 848798 N/A N/A GGGAAGAAGCTTCCCA  0 4882  4897 780 848814 N/A N/A AGAAAGTCAAAGGCTC 74  5360  5375 781848830 N/A N/A GACACCATCATCGCTG 67  5836  5851 782 848846 N/A N/AGGCAGACATACCTGCT  0  6694  6709 783 848862 N/A N/A GGCCTTAAGCTGCTTT 10 7048  7063 784 848878 N/A N/A ATATAAATCTCCCATC  9  7281  7296 785848894 N/A N/A TCACATGGTTATATAA 37  7640  7655 786 848271  667  682TATCCCCGGCGGGCAG 35  7940  7955 787 848283  683  698 CAGGATCTTGGTGAGG 49 7956  7971 788 848298  740  755 CAGCAGGTCGCCACTC  5  8013  8028 789848910 N/A N/A AGCAATGGGCCTACTA  0  8107  8122 790 848926 N/A N/AAGCAACAGCTTCAAAG 11  8453  8468 791 848942 N/A N/A CTGATTAACCCATGGG 46 8833  8848 792 848958 N/A N/A TTACAGATAGAGGAAT  0  9365  9380 793848974 N/A N/A GGACATGGAGTGAAGC  7 10200 10215 794 848313  801  816GGGCAAAGACAGAGGA 11 10562 10577 795 848990 N/A N/A GGTAGAGTCACCATCA 4010953 10968 796 849006 N/A N/A TCTCACAGCAACCTGT  0 11548 11563 797849022 N/A N/A TCCTAACCCCCACAAC  0 12373 12388 798 849038 N/A N/ATGCAGGGCTAAAATCA 34 12880 12895 799 849044 N/A N/A GGATACACAGGCTCGC 7813108 13123 337 849054 N/A N/A GGCAGGTGGGCCTGGT  0 13689 13704 800849070 N/A N/A GGATACATTGGCAGAC 41 14123 14138 801 849086 N/A N/AGTGTTAGGAGCTTTCA 60 14465 14480 802 849102 N/A N/A AGACACATCACAGGAC 2714918 14933 803 849118 N/A N/A AAGAAGCAGGCACTGG 32 15608 15623 804849134 N/A N/A AAGGAAAGGGAGGCCT  0 16081 16096 805 848328  964  979TCGAAGTCGGTGACCA 17 16356 16371 806 848344  999 1014 TGTGGAAGCGGGTCCC 2416391 16406 807 848358 1055 1070 GCCGCTGACCACCCCT  0 16685 16700 808468460 1075 1090 TTGGCCACGCCGGCAT 30 16705 16720  48 848373 1079 1094ACCCTTGGCCACGCCG 30 16709 16724 809 848389 1145 1160 TATGAGGGTGCCGCTA  416775 16790 810 849150 N/A N/A ACTTACCTATGAGGGT  0 16782 16797 811849166 N/A N/A GGCTTACAGTGGAAGT  8 17558 17573 812 849182 N/A N/ATGCCCCGTGCCAGGTC 41 17961 17976 813 849198 N/A N/A CCAGGAGCCGTGGCCA 1218537 18552 814 849214 N/A N/A ACACAAACTTGAACAG 10 19249 19264 815849230 N/A N/A TCTTAATATATACATC  0 19567 19582 816 849246 N/A N/AGAACAAAGCCAAGGTC  0 19970 19985 817 848405 1286 1301 GGTGACCAGCACGACC  020117 20132 818 849262 N/A N/A CCTTGATAGGCCAGGG  0 20937 20952 819849278 N/A N/A TCCCACTCAAATGTCC  0 21205 21220 820 848421 1425 1440CCACACAGCGGCCAAA  0 21397 21412 821 848437 1554 1569 CGGCAGACAGCATCAT 7222047 22062 822 848453 1681 1696 GGCAGGGCGGCCACCA  0 22174 22189 823849294 N/A N/A AAAATATTGCACAGCC  0 22353 22368 824 848469 1760 1775GGCCATCCGTGTAGGC  0 22542 22557 825 849310 N/A N/A AGCATTAAGACCCCAT 2123167 23182 826 848484 1929 1944 GGCAGCACCTGGCAAT 17 23549 23564 827848499 2000 2015 GTGGACACGGGTCCCC 17 23620 23635 828 849326 N/A N/AAGAAAGAGACCCCTCC  0 23692 23707 829 849342 N/A N/A AAACAATAGTAACGAT  024153 24168 830 849358 N/A N/A TCTGAACATGGTAGGG 58 24790 24805 831849374 N/A N/A GCCCATCCCCATCAGA  6 25303 25318 832 848515 2157 2172GGGCATGGCAGCAGGA 17 25488 25503 833 849390 N/A N/A CTTCAGCATGCAGCTC  626093 26108 834 849406 N/A N/A TGAAACTGATGGCCCC 22 27070 27085 835848531 2383 2398 CAGCAGATGGCAACGG 25 27526 27541 836 848547 2522 2537GGCCATGGAGGGCTGA  0 27665 27680 837 848563 2623 2638 AGGCACCCAGAGTGAG 1227766 27781 838 848579 2716 2731 AGCTGGCAGCACCCGA 20 27859 27874 839848594 2770 2785 GAACAAGAGCTCAATA 13 27913 27928 840 848610 2822 2837GGGAAGAATCCTGCCT 25 27965 27980 841 848626 2948 2963 CATTAATCAGGGAGCC 2128091 28106 842 848641 3052 3067 GAGCAGAGTAAAGGTG 23 28195 28210 843848656 3126 3141 CACACTGCCGAGTCAG 56 28269 28284 844 848672 3213 3228AGGTTTCTTCCTCTGT 60 28356 28371 845 848687 3243 3258 GAGCTTGGCAGGCACG 6728386 28401 846 848703 3266 3281 TTCTGGCTCAGTTCCT 20 28409 28424 847848719 3334 3349 GTAAAAATGAGGAGCC 29 28477 28492 848 848735 3421 3436AAAAAGTTCCATGCCT  9 28564 28579 849 848751 3506 3521 GGAGGGACAGTTGTTG  028649 28664 850 848766 3590 3605 TAGAAAAGTTGGCTGT 22 28733 28748 851

TABLE 12Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 848345 1012 1027TTGCTGGCCTGTCTGT  3 N/A N/A 852 848390 1148 1163 GCCTATGAGGGTGCCG  0 N/AN/A 853 848454 1708 1723 TGCCAACCTGCCCCAT  8 N/A N/A 854 848210   56  71 CGGAAACCTTCTAGGG  0  3531  3546 855 848226  244  259AGCTGAAGTTCAGGAG 50  3719  3734 856 848242  480  495 AGGCTAGCACCAGCTC 37 3955  3970 857 848257  552  567 TGGCGCAGCGGTGGAA  0  4027  4042 858848783 N/A N/A CCACTACCCGTCCTCC  0  4211  4226 859 848799 N/A N/ACGGGAAGAAGCTTCCC  0  4883  4898 860 848815 N/A N/A CCCCACAACATCCCTC  5 5377  5392 861 848831 N/A N/A AAAGAACTGTGGACAT 43  5935  5950 862848847 N/A N/A AGGCAGACATACCTGC  0  6695  6710 863 848863 N/A N/ACTGCAAGCTCTCCAGG 17  7095  7110 864 848879 N/A N/A CAAGAGTGACGGTTAT 45 7295  7310 865 848895 N/A N/A TATTATTCACATGGTT 41  7646  7661 866848272  668  683 GTATCCCCGGCGGGCA 37  7941  7956 867 848284  684  699GCAGGATCTTGGTGAG  0  7957  7972 868 848299  743  758 CTCCAGCAGGTCGCCA 20 8016  8031 869 848911 N/A N/A ATACATACTTGCTGTC 21  8137  8152 870848927 N/A N/A TGGATAGCAACAGCTT 70  8458  8473 871 848943 N/A N/ATGCTGAGCTGATTAAC 27  8840  8855 872 848959 N/A N/A AATTATTATAACTGGT  0 9381  9396 873 848975 N/A N/A GGGCAGGATGGACATG  4 10209 10224 874848314  817  832 TTCCACGGGATGCTCT  3 10578 10593 875 848991 N/A N/ATTAAAGGATCTGGTCC  3 11011 11026 876 849007 N/A N/A GTGTATGCAAAGTCAC 5511624 11639 877 849023 N/A N/A AGGGAACAGTGGCTGC 42 12407 12422 878849039 N/A N/A TTATTGAATGGTAAGA 37 12905 12920 879 849044 N/A N/AGGATACACAGGCTCGC 77 13108 13123 337 849055 N/A N/A CTGCAAGCATGGCCAG 3313710 13725 880 849071 N/A N/A ACAACTGGATACATTG 57 14129 14144 881849087 N/A N/A CACTTTGGTTTCTTCT 28 14560 14575 882 849103 N/A N/ACTTATGGCTTCGGTCA 42 15107 15122 883 849119 N/A N/A AGATACCAGGAGGGCT 4815730 15745 884 849135 N/A N/A ATAGACAAGGAAAGGG 68 16087 16102 885848329  971  986 CACATTCTCGAAGTCG 25 16363 16378 886 848359 1056 1071GGCCGCTGACCACCCC 19 16686 16701 887 468460 1075 1090 TTGGCCACGCCGGCAT 1016705 16720  48 848374 1080 1095 CACCCTTGGCCACGCC 37 16710 16725 888849151 N/A N/A ATGGAGAGAGACCAGC 20 16811 16826 889 849167 N/A N/ACACCAGAGCCAGTGTT  0 17590 17605 890 849183 N/A N/A CTGCCCCGTGCCAGGT  017962 17977 891 849199 N/A N/A TGTCAAATGAGGTGTG 15 18586 18601 892849215 N/A N/A ATGAGAAGGGCACACT  0 19342 19357 893 849231 N/A N/AAACTCTTAATATATAC  0 19570 19585 894 849247 N/A N/A AGGAACAAAGCCAAGG  319972 19987 895 848406 1293 1308 CGGCAGCGGTGACCAG  0 20124 20139 896849263 N/A N/A GCCTTGATAGGCCAGG 33 20938 20953 897 849279 N/A N/AAGAGAAAGGAGCCCAA  0 21290 21305 898 848422 1427 1442 GTCCACACAGCGGCCA  521399 21414 899 848438 1558 1573 GGCTCGGCAGACAGCA 58 22051 22066 900849295 N/A N/A GGCTTAAAGAACATAC 28 22397 22412 901 848470 1772 1787GGCGACGGCTGTGGCC  0 22554 22569 902 849311 N/A N/A GAGCATTAAGACCCCA 3223168 23183 903 468480 1930 1945 AGGCAGCACCTGGCAA 19 23550 23565 904848500 2001 2016 AGTGGACACGGGTCCC 56 23621 23636 905 849327 N/A N/ATCACACTTGTGAGGAC  0 23737 23752 906 849343 N/A N/A AAAGCAACGGGTGATG  024170 24185 907 849359 N/A N/A GTAAGATGGAAAGAGA 55 24877 24892 908849375 N/A N/A CTGGGATGCTCCGTCT  0 25324 25339 909 848516 2172 2187TGCATTCCAGACCTGG  6 25503 25518 910 849391 N/A N/A TGCTAACAACCTTCAG  026103 26118 911 849407 N/A N/A TACCATGCCAGTGCCA 29 27098 27113 912848532 2386 2401 CGGCAGCAGATGGCAA 27 27529 27544 913 848548 2527 2542TGCCAGGCCATGGAGG  0 27670 27685 914 848564 2628 2643 GGAGGAGGCACCCAGA 2027771 27786 915 468487 2720 2735 GAGCAGCTGGCAGCAC 42 27863 27878 916848595 2771 2786 GGAACAAGAGCTCAAT 47 27914 27929 917 848611 2828 2843ATCCATGGGAAGAATC 15 27971 27986 918 848627 2951 2966 CTCCATTAATCAGGGA 2628094 28109 919 848642 3055 3070 ATAGAGCAGAGTAAAG  0 28198 28213 920848657 3127 3142 GCACACTGCCGAGTCA 46 28270 28285 921 848673 3214 3229CAGGTTTCTTCCTCTG 34 28357 28372 922 848688 3245 3260 GTGAGCTTGGCAGGCA 7028388 28403 923 848704 3267 3282 TTTCTGGCTCAGTTCC 54 28410 28425 924848720 3336 3351 CCGTAAAAATGAGGAG 17 28479 28494 925 848736 3423 3438GGAAAAAGTTCCATGC 57 28566 28581 926 468502 3508 3523 AAGGAGGGACAGTIGT  028651 28666 927 848767 3592 3607 TCTAGAAAAGTTGGCT 39 28735 28750 928

TABLE 13Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 848258  561  576ACGGATCCTTGGCGCA 38 N/A N/A  929 848300  753  768 TCAAGGCCAGCTCCAG 23N/A N/A  930 848346 1014 1029 ACTTGCTGGCCTGTCT  0 N/A N/A  931 8483911154 1169 CTCCAGGCCTATGAGG  0 N/A N/A  932 848211   58   73TGCGGAAACCTTCTAG  0 3533  3548  933 848227  258  273 GAGGACTGTGCAGGAG 42 3733  3748  934 848243  481  496 AAGGCTAGCACCAGCT 23  3956  3971  935848784 N/A N/A GGATATCCTGGCAGTG  0  4350  4365  936 848800 N/A N/AGAAAAAGCTAGTGGTC 30  4905  4920  937 848816 N/A N/A CTTGAAATGCCCTTTC  0 5408  5423  938 848832 N/A N/A TACATTTCAGACGGTG 61  6279  6294  939848848 N/A N/A ATTATAGCAGCCACTA 13  6761  6776  940 848864 N/A N/AATTTATTCAGCTCATG  1  7114  7129  941 848880 N/A N/A TGGGAAAGTCAAGAGT 44 7304  7319  942 848896 N/A N/A GCCAACTATTATTATT 26  7655  7670  943466846  669  684 GGTATCCCCGGCGGGC 45  7942  7957  944 848285  685  700TGCAGGATCTTGGTGA  0  7958  7973  945 848912 N/A N/A GATAAGTGCTCAATAC 33 8149  8164  946 848928 N/A N/A CTGGATAGCAACAGCT 71  8459  8474  947848944 N/A N/A CGCCACAGGCCTTGGT 30  8881  8896  948 848960 N/A N/ACCAATTATTATAACTG 21  9383  9398  949 848976 N/A N/A AGCCAGGCTGTGCCAA  010231 10246  950 848315  824  839 CTCCAGGTTCCACGGG  0 10585 10600  951848992 N/A N/A ATTAAAGGATCTGGTC  2 11012 11027  952 849008 N/A N/ACAGACATATGCAAGGT 68 11652 11667  953 849024 N/A N/A GCCCAAGGGAGAGGAG  012421 12436  954 849040 N/A N/A GTTATTATTGAATGGT 80 12909 12924  955849044 N/A N/A GGATACACAGGCTCGC 76 13108 13123  337 849056 N/A N/ACAGGAGGCTGCAAGCA 49 13717 13732  956 849072 N/A N/A CAGGAAGCAGCCCAAG  014175 14190  957 849088 N/A N/A CTCCACTGATCAGTCC 49 14582 14597  958849104 N/A N/A CCAAATATGCTGCAGA 20 15185 15200  959 849120 N/A N/AAGAGACAGGAAGCTGC 63 15808 15823  960 849136 N/A N/A CACAGAAACTACAGAG  016199 16214  961 848330  974  989 GGGCACATTCTCGAAG  0 16366 16381  962848360 1057 1072 CGGCCGCTGACCACCC  0 16687 16702  963 468460 1075 1090TTGGCCACGCCGGCAT  0 16705 16720   48 848375 1081 1096 GCACCCTTGGCCACGC24 16711 16726  964 849152 N/A N/A CCAGATGGAGAGAGAC 26 16815 16830  965849168 N/A N/A GGCTAGTGGGCTGCCT  0 17613 17628  966 849184 N/A N/ACCCTGCCCCGTGCCAG  0 17964 17979  967 849200 N/A N/A GCCTAACGCAGCTCTG 6218644 18659  968 849216 N/A N/A GAGGAATGAGAAGGGC 61 19347 19362  969849232 N/A N/A CCGCACGGTCACATGA 19 19592 19607  970 849248 N/A N/AGTGCCACAAGAAGCCC  9 20268 20283  971 20286 20301 848407 1301 1316GAAGTTGCCGGCAGCG 24 20132 20147  972 849264 N/A N/A TAAATTCGGCCGCCAG  920958 20973  973 848423 1433 1448 AAAGAGGTCCACACAG  0 21405 21420  974849280 N/A N/A CTGTTAGCATCACGGT 25 21542 21557  975 848439 1574 1589GGCCAGGGTGAGCTCC  0 22067 22082  976 849296 N/A N/A GAGAGAGGAGGGCTTA  022407 22422  977 848455 1716 1731 AAAACAGCTGCCAACC  0 22498 22513  978848471 1774 1789 CGGGCGACGGCTGTGG  0 22556 22571  979 849312 N/A N/AACACGAGCATTAAGAC 45 23172 23187  980 848485 1932 1947 GCAGGCAGCACCTGGC 7 23552 23567  981 848501 2002 2017 CAGTGGACACGGGTCC 37 23622 23637 982 849328 N/A N/A CTGCAAGTCAGGCTTG 20 23764 23779  983 849344 N/A N/ATTTAAAGCAACGGGTG 47 24173 24188  984 849360 N/A N/A AGCTACACTGAGGCTC 2324906 24921  985 848517 2177 2192 GACTTTGCATTCCAGA 41 25508 25523  986849376 N/A N/A GGCAGAGGAAAGCCAG  0 25355 25370  987 849392 N/A N/ATGTCACATTCCAGGGC 43 26171 26186  988 849408 N/A N/A AGATAGACAGATGCCT 827113 27128  989 848533 2402 2417 CGCCAGGTGCCGGCTC 0 27545 27560  990848549 2550 2565 GCGGAAGCATCCCCAT 0 27693 27708  991 848565 2669 2684CCCCACAGTGAGGGAG 20 27812 27827  992 848580 2727 2742 ACATTGGGAGCAGCTG34 27870 27885  993 848596 2772 2787 CGGAACAAGAGCTCAA 70 27915 27930 994 848612 2832 2847 CCCTATCCATGGGAAG 39 27975 27990  995 848628 29592974 AGCTAAGCCTCCATTA 15 28102 28117  996 848643 3057 3072GCATAGAGCAGAGTAA  0 28200 28215  997 848658 3128 3143 TGCACACTGCCGAGTC49 28271 28286  998 848674 3215 3230 CCAGGTTTCTTCCTCT 58 28358 28373 999 848689 3246 3261 TGTGAGCTTGGCAGGC 39 28389 28404 1000 848705 32683283 GTTTCTGGCTCAGTTC  3 28411 28426 1001 848721 3338 3353ACCCGTAAAAATGAGG  0 28481 28496 1002 848737 3425 3440 ACGGAAAAAGTTCCAT18 28568 28583 1003 848752 3511 3526 CTCAAGGAGGGACAGT 33 28654 286691004 848768 3599 3614 AAACAGGTCTAGAAAA 17 28742 28757 1005

TABLE 14Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ SEQ SEQ SEQ ID: 1 ID: 1 ID: 2 ID 2: Start Stop % Start Stop SEQISIS No Site Site Sequence inhibition Site Site ID NO 468456 1015 1030CACTTGCTGGCCTGTC 22 N/A N/A 1006 848259  565  580 CTCCACGGATCCTTGG  0N/A N/A 1007 848301  754  769 TTCAAGGCCAGCTCCA 42 N/A N/A 1008 8483921159 1174 ATAAACTCCAGGCCTA 37 N/A N/A 1009 848212   64   79CGTCGCTGCGGAAACC 41  3539  3554 1010 848228  293  308 GTCCACGCCGGCGGCG32  3768  3783 1011 848244  482  497 CAAGGCTAGCACCAGC 45  3957  39721012 848785 N/A N/A GAAATCTGGGCAGGAT 39  4362  4377 1013 848801 N/A N/ACACCACAGCTAGTGAG 28  5003  5018 1014 848817 N/A N/A CACTATTTCCAGAACA 20 5445  5460 1015 848833 N/A N/A AATAATCTCATGTCAG 77  6356  6371 1016848849 N/A N/A CAAATTATAGCAGCCA 85  6764  6779 1017 848865 N/A N/AGATTTATTCAGCTCAT 65  7115  7130 1018 848881 N/A N/A CCACAGTACCTATGAC  0 7330  7345 1019 848897 N/A N/A CAAATGCGGACCAAAA 38  7694  7709 1020848273  670  685 AGGTATCCCCGGCGGG 26  7943  7958 1021 848286  686  701ATGCAGGATCTTGGTG  0  7959  7974 1022 848913 N/A N/A CGATAAGTGCTCAATA 25 8150  8165 1023 848929 N/A N/A AAAGAAGGGACTTCAC 29  8476  8491 1024848945 N/A N/A ACTTAGACTACGGGTT 25  8931  8946 1025 848961 N/A N/ATCCTTAAATGAATGTT 41  9399  9414 1026 848977 N/A N/A CTTGATCAGGCTGGGA  010332 10347 1027 848316  833  848 GGTAATCCGCTCCAGG 59 10594 10609 1028848993 N/A N/A TCCCATTAAAGGATCT 23 11016 1103 1029 849009 N/A N/AACACATGACCGACCAG  0 11773 11788 1030 849025 N/A N/A CTCCAGCCAAGCCCTT 4012570 12585 1031 849041 N/A N/A TCTGGATACACTGTTG 27 13008 13023 1032849044 N/A N/A GGATACACAGGCTCGC 85 13108 13123  337 849057 N/A N/AGGTCAGGAGGCTGCAA 61 13720 13735 1033 849073 N/A N/A TGCTCAGGAAGCAGCC 4314179 14194 1034 849089 N/A N/A CCCTAAGGCCTCCAGT 41 14610 14625 1035849105 N/A N/A CTGAACAGCACCTCTG 37 15220 15235 1036 849121 N/A N/ATTAGAGACAGGAAGCT 28 15810 15825 1037 849137 N/A N/A CTTATAGTTAACACAC  016212 16227 1038 848331  980  995 CTCCTCGGGCACATTC  7 16372 16387 1039848361 1058 1073 CCGGCCGCTGACCACC 30 16688 16703 1040 468460 1075 1090TTGGCCACGCCGGCAT 31 16705 16720   48 848376 1082 1097 GGCACCCTTGGCCACG52 16712 16727 1041 849153 N/A N/A GCCCAAGCCACCTCCC  0 16840 16855 1042849169 N/A N/A AGGAAAGTCTCAGGGC 78 17681 17696 1043 849185 N/A N/AAGCCCCTGCCCCGTGC 18 17967 17982 1044 849201 N/A N/A GGCCTAACGCAGCTCT 3418645 18660 1045 849217 N/A N/A AGGCACCAAGAGGATG 20 19370 19385 1046849233 N/A N/A GCGGATTTCAGACTTG 80 19613 19628 1047 848408 1304 1319CCGGAAGTTGCCGGCA  3 20135 20150 1048 849249 N/A N/A CGTGCCACAAGAAGCC  220269 20284 1049 20287 20302 849265 N/A N/A GCCTTTAAATTCGGCC  0 2096320978 1050 848424 1435 1450 GCAAAGAGGTCCACAC  0 21407 21422 1051 849281N/A N/A GGCACTGGAGGTCCCG 31 21583 21598 1052 848440 1582 1597CTCAACTCGGCCAGGG 43 22075 22090 1053 849297 N/A N/A TAGGAGAGAGGAGGGC 2122410 22425 1054 848456 1718 1733 GCAAAACAGCTGCCAA 24 22500 22515 1055848472 1778 1793 GCAGCGGGCGACGGCT  0 22560 22575 1056 849313 N/A N/ACTCTCACACGAGCATT 11 23177 23192 1057 848486 1933 1948 AGCAGGCAGCACCTGG35 23553 23568 1058 848502 2003 2018 GCAGTGGACACGGGTC 43 23623 236381059 849329 N/A N/A GTATGGAACTGCAAGT  0 23772 23787 1060 849345 N/A N/AATTTTAAAGCAACGGG 75 24175 24190 1061 849361 N/A N/A AAACCTAAAATAGTGG  424928 24943 1062 849377 N/A N/A GGGCAGAGGAAAGCCA 12 25356 25371 1063848518 2190 2205 TTCCATGCTCCTTGAC 11 25521 25536 1064 849393 N/A N/AATACACTCAGGTTTCT 45 26199 26214 1065 849409 N/A N/A ACCCAGGCGGTTCTGC 1027185 27200 1066 848534 2427 2442 GTCACTGGAGCTCCTG 59 27570 27585 1067848550 2554 2569 AAAGGCGGAAGCATCC 23 27697 27712 1068 848566 2677 2692GTGAAATGCCCCACAG 34 27820 27835 1069 848581 2729 2744 GCACATTGGGAGCAGC58 27872 27887 1070 848597 2773 2788 ACGGAACAAGAGCTCA 77 27916 279311071 848613 2869 2884 CAACGATGTTTGTCCC 67 28012 28027 1072 848629 29622977 GAAAGCTAAGCCTCCA 69 28105 28120 1073 848644 3059 3074TGGCATAGAGCAGAGT 49 28202 28217 1074 848659 3129 3144 CTGCACACTGCCGAGT48 28272 28287 1075 848675 3216 3231 TCCAGGTTTCTTCCTC 63 28359 283741076 848690 3247 3262 GTGTGAGCTTGGCAGG 78 28390 28405 1077 848706 32693284 CGTTTCTGGCTCAGTT 55 28412 28427 1078 848722 3342 3357TGTTACCCGTAAAAAT  0 28485 28500 1079 848738 3427 3442 TAACGGAAAAAGTTCC64 28570 28585 1080 848753 3513 3528 TGCTCAAGGAGGGACA 27 28656 286711081 848769 3601 3616 CAAAACAGGTCTAGAA 43 28744 28759 1082

TABLE 15Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ ID: 1 SEQ ID: 1 % SEQ ID: 2 SEQ ID: 2 SEQ ISIS No Start SiteStop Site Sequence inhibition Start Site Stop Site ID NO 848302  755 770 CTTCAAGGCCAGCTCC  0 N/A N/A 1083 848347 1016 1031 ACACTTGCTGGCCTGT 0 N/A N/A 1084 848213   85  100 CTGCAACCATGAGCGC  4  3560  3575 1085848229  309  324 CTAGAGGCCGTGCGCG 18  3784  3799 1086 848245 483  498GCAAGGCTAGCACCAG 45  3958  3973 1087 848786 N/A N/A GAAACTGGGAAATCTG  0 4370  4385 1088 848802 N/A N/A GTCAAGCACCACAGCT 54  5009  5024 1089848818 N/A N/A CCTCAAGGGCTTGGTT  0  5509  5524 1090 848834 N/A N/AAATCTATGCAGCAAAA  0  6407  6422 1091 848850 N/A N/A AACAAATTATAGCAGC 75 6766  6781 1092 848882 N/A N/A GACCACAGTACCTATG 30  7332  7347 1093848898 N/A N/A ACCAAATGCGGACCAA 66  7696  7711 1094 848260  571  586GGCAACCTCCACGGAT 53  7844  7859 1095 466847  671  686 GAGGTATCCCCGGCGG73  7944  7959 1096 848287  687  702 CATGCAGGATCTTGGT  5  7960  79751097 848914 N/A N/A TGCTTGGTACCCGATA 39  8161  8176 1098 848930 N/A N/ACCTAAAGAAGGGACTT 18  8479  8494 1099 848946 N/A N/A AGGCATTGACTTGTCA 46 8956  8971 1100 848962 N/A N/A TACCTAGCATCTGCTG  0  9437  9452 1101848978 N/A N/A ACAGACTAGGAGCCTG  0 10361 10376 1102 848317  852  867CCGCCCGGTACCGTGG  0 10613 10628 1103 848994 N/A N/A AGGCATCCCAGACAGG 5211042 11057 1104 849010 N/A N/A GCACACATGACCGACC  0 11775 11790 1105849026 N/A N/A CCCCATGCCAGCCCAA 33 12604 12619 1106 849042 N/A N/AATTAATCTTCTGGATA 13 13016 13031 1107 849044 N/A N/A GGATACACAGGCTCGC 8213108 13123  337 849058 N/A N/A GGACAGGGTCAGGAGG 22 13726 13741 1108849074 N/A N/A GGATACAGGTGCTCAG 55 14188 14203 1109 849090 N/A N/AACACACACTGTCTACC 38 14629 14644 1110 849106 N/A N/A CCTCAGGTGGAATCAG 5615306 15321 1111 849122 N/A N/A GAATAACAGTGATGTC 17 15852 15867 1112849138 N/A N/A ACCTTATAGTTAACAC  4 16214 16229 1113 848332  982  997TCCTCCTCGGGCACAT 21 16374 16389 1114 848362 1059 1074 CCCGGCCGCTGACCAC 0 16689 16704 1115 468460 1075 1090 TTGGCCACGCCGGCAT  0 16705 16720  48 848377 1083 1098 TGGCACCCTTGGCCAC  0 16713 16728 1116 849154 N/AN/A GGTCAAGGCTGAACTC 27 17014 17029 1117 849170 N/A N/A TAGGAAAGTCTCAGGG36 17682 17697 1118 849186 N/A N/A AGGCAATAGTGACTGT 52 17985 18000 1119849202 N/A N/A CTGGAGACTGGAGGCC  0 18657 18672 1120 849218 N/A N/AGAAAGAGAGGCACCAA  0 19377 19392 1121 849234 N/A N/A TGCGGATTTCAGACTT 2419614 19629 1122 848393 1161 1176 GAATAAACTCCAGGCC  0  1999 20007 1123848409 1322 1337 GTAGAGGCAGGCATCG 42 20153 20168 1124 849250 N/A N/ACAGAAGGGTTCGGCCT 32 20310 20325 1125 849266 N/A N/A GGCTTTGTTTGCTTGA 6820980 20995 1126 848425 1437 1452 GGGCAAAGAGGTCCAC  0 21409 21424 1127849282 N/A N/A CATCAGGCCTACTTCA  0 21624 21639 1128 848441 1603 1618GAGAAGTGGATCAGTC  0 22096 22111 1129 849298 N/A N/A TGCAAAAAGGGCCTGG  022482 22497 1130 848457 1720 1735 CTGCAAAACAGCTGCC 13 22502 22517 1131848473 1780 1795 GCGCAGCGGGCGACGG  0 22562 22577 1132 849314 N/A N/AATCCAGCCAGCTCCAC  7 23207 23222 1133 848487 1934 1949 TAGCAGGCAGCACCTG 0 23554 23569 1134 848503 2004 2019 GGCAGTGGACACGGGT 26 23624 236391135 849330 N/A N/A GGCAGAACCAGAGTAT  0 23784 23799 1136 849346 N/A N/ACAATAGTTGCCTATAC  0 24201 24216 1137 849362 N/A N/A AGACACACCCATTGGC  824948 24963 1138 848519 2198 2213 GGCCGGGATTCCATGC  0 25529 25544 1139849378 N/A N/A ACGCAGCACCCCACCC  0 25581 25596 1140 849394 N/A N/AAAGGAGAGTTATACAC 53 26209 26224 1141 849410 N/A N/A GGACACAACCGTGTAT  027220 27235 1142 848535 2447 2462 ACCCATCCTGGGATGG  0 27590 27605 1143848551 2556 2571 GGAAAGGCGGAAGCAT 38 27699 27714 1144 848567 2684 2699TTGAATGGTGAAATGC 65 27827 27842 1145 848582 2731 2746 CGGCACATTGGGAGCA33 27874 27889 1146 848598 2774 2789 CACGGAACAAGAGCTC 68 27917 279321147 848614 2872 2887 CCCCAACGATGTTTGT 32 28015 28030 1148 848630 29642979 CAGAAAGCTAAGCCTC 72 28107 28122 1149 466853 3067 3082GCACAGCCTGGCATAG 51 28210 28225 1150 848660 3130 3145 ACTGCACACTGCCGAG 1 28273 28288 1151 848676 3218 3233 GTTCCAGGTTTCTTCC 43 28361 283761152 848691 3248 3263 TGTGTGAGCTTGGCAG 22 28391 28406 1153 848707 32843299 AGCCAGCCCAATCTGC  0 28427 28442 1154 848723 3348 3363CCTCACTGTTACCCGT 36 28491 28506 1155 848739 3429 3444 GATAACGGAAAAAGTT 0 28572 28587 1156 848754 3543 3558 ATAAATGTCTGCTTGC 52 28686 287011157 848770 3603 3618 AGCAAAACAGGTCTAG 60 28746 28761 1158

TABLE 16Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ ID: 1 SEQ ID: 1 % SEQ ID: 2 SEQ ID: 2 SEQ ISIS No Start SiteStop Site Sequence inhibition Start Site Stop Site ID NO 848303  756 771 ACTTCAAGGCCAGCTC 43 N/A N/A 1159 848214  105  120 AACTGAACGGCGGCGC 0  3580  3595 1160 848230  311  326 ACCTAGAGGCCGTGCG 30  3786  38011161 848246  484  499 CGCAAGGCTAGCACCA 45  3959  3974 1162 848787 N/AN/A GGCGAGGCAGAAACTG 29  4379  4394 1163 848803 N/A N/A TCAAGGATAAGTGACT42  5026  5041 1164 848819 N/A N/A AGAAACATCCTCAAGG 38  5517  5532 1165848835 N/A N/A TAATCTATGCAGCAAA  0  6408  6423 1166 848851 N/A N/ACCAAATTTCCACATGA 41  6797  6812 1167 848867 N/A N/A AAGGAATACCTGAAGG 22 7153  7168 1168 848883 N/A N/A CCTCAGATACCTCTGC 17  7359  7374 1169848899 N/A N/A GTATAGTAGATGATAA  0  7719  7734 1170 848261  576  591TGCCAGGCAACCTCCA 14  7849  7864 1171 848274  672  687 TGAGGTATCCCCGGCG14  7945  7960 1172 848288  688  703 ACATGCAGGATCTTGG 16  7961  79761173 848915 N/A N/A AGCCAGTAGTTACTGT  9  8176  8191 1174 848931 N/A N/AGAAGATGTGACATCCA 28  8517  8532 1175 848947 N/A N/A GAGCAGACTGATGGAA  4 8971  8986 1176 848963 N/A N/A GTGGTAACAGCCTCCT 26  9526  9541 1177848979 N/A N/A GGACAGACTAGGAGCC  4 10363 10378 1178 848318  858  873ATTCATCCGCCCGGTA  0 10619 10634 1179 848995 N/A N/A AGGTAGGCAGAGGCAT 2011052 11067 1180 849011 N/A N/A GGCCGGGTCAGCACAC  0 11785 11800 1181849027 N/A N/A GGCCCATGCTTGTGGC  0 12663 12678 1182 849043 N/A N/AAGGAATGGATTAATCT 48 13024 13039 1183 849044 N/A N/A GGATACACAGGCTCGC 8113108 13123  337 849059 N/A N/A TCTAAGTGCAGGCGGT 23 13776 13791 1184849075 N/A N/A CAGCAAGGTGGGCAGA 10 14274 14289 1185 849091 N/A N/AGCACAGTCCGAACTGT  0 14660 14675 1186 849107 N/A N/A GCGGAAATGGCCTGGC  015353 15368 1187 849123 N/A N/A GGAGAATAACAGTGAT 62 15855 15870 1188849139 N/A N/A AACCTTATAGTTAACA  0 16215 16230 1189 848333  983  998GTCCTCCTCGGGCACA 18 16375 16390 1190 468457 1017 1032 CACACTTGCTGGCCTG 0 16647 16662 1191 848363 1060 1075 TCCCGGCCGCTGACCA 30 16690 167051192 468460 1075 1090 TTGGCCACGCCGGCAT  0 16705 16720   48 848378 10841099 CTGGCACCCTTGGCCA  0 16714 16729 1193 849155 N/A N/AAGACATCACCGGATTT 42 17074 17089 1194 849171 N/A N/A CTGTAGGAAAGTCTCA 4917685 17700 1195 849187 N/A N/A GGACACACAATCACCT 14 18033 18048 1196849203 N/A N/A CAGTAACAGTTCTAAC  0 18706 18721 1197 849219 N/A N/ACTTATTATCCCTTTCC 29 19395 19410 1198 849235 N/A N/A TCAAATTCTGCAGGAA  019653 19668 1199 848394 1163 1178 CCGAATAAACTCCAGG 42 19994 20009 1200848410 1324 1339 GAGTAGAGGCAGGCAT  0 20155 20170 1201 849251 N/A N/ACCAAAGCCAGAAGGGT 32 20317 20332 1202 849267 N/A N/A TTCTAAGTGCCACGGG 5621029 21044 1203 848426 1455 1470 CAATGATGTCCTCCCC  0 21427 21442 1204849283 N/A N/A TCCCGATCAAATGTCC  0 21772 21787 1205 848442 1606 1621GCAGAGAAGTGGATCA  0 22099 22114 1206 849299 N/A N/A TGCCAACCTGCAAAAA  022490 22505 1207 848458 1728 1743 ATACAGTCCTGCAAAA  0 22510 22525 1208848474 1803 1818 AGCTCAGCAGCTCCTC 11 22585 22600 1209 849315 N/A N/AATCCAGGAGGCCAAAG  0 23228 23243 1210 848488 1937 1952 GGGTAGCAGGCAGCAC 0 23557 23572 1211 848504 2005 2020 TGGCAGTGGACACGGG 17 23625 236401212 849331 N/A N/A GGAGAAGTAAGGTCAC 52 23827 23842 1213 849347 N/A N/AGTCAATAGTTGCCTAT 13 24203 24218 1214 849363 N/A N/A GTTCAAGGGTAAGCCG 2525011 25026 1215 849379 N/A N/A TGGCACAAACTGACAC  0 25619 25634 1216849395 N/A N/A TTAAGAGTGGACTCCT 10 26260 26275 1217 849411 N/A N/ACCAAAGTGCAGACGGC  0 27260 27275 1218 848520 2222 2237 GGCCACGGTCACCTGC 0 27365 27380 1219 848536 2455 2470 CCCCAGACACCCATCC 26 27598 276131220 848552 2558 2573 CCGGAAAGGCGGAAGC  0 27701 27716 1221 848568 27002715 GCACAGCTCGACCTGT  3 27843 27858 1222 848583 2737 2752GGACATCGGCACATTG 68 27880 27895 1223 848599 2775 2790 GCACGGAACAAGAGCT19 27918 27933 1224 848615 2885 2900 TTTCACACTCACCCCC  7 28028 280431225 848631 2967 2982 ATCCAGAAAGCTAAGC  1 28110 28125 1226 848645 30723087 TGCTAGCACAGCCTGG 19 28215 28230 1227 848661 3131 3146CACTGCACACTGCCGA 11 28274 28289 1228 848677 3219 3234 GGTTCCAGGTTTCTTC61 28362 28377 1229 848692 3249 3264 CTGTGTGAGCTTGGCA 69 28392 284071230 848708 3288 3303 TCAGAGCCAGCCCAAT  8 28431 28446 1231 848724 33783393 GAGCTTCCTGGTCTGT 47 28521 28536 1232 848740 3431 3446GTGATAACGGAAAAAG 65 28574 28589 1233 848755 3545 3560 AGATAAATGTCTGCTT14 28688 28703 1234 848771 3616 3631 TTCAAGTTACAAAAGC 57 28759 287741235

TABLE 17Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ ID: 1 SEQ ID: 1 % SEQ ID: 2 SEQ ID: 2 SEQ ISIS No Start SiteStop Site Sequence inhibition Start Site Stop Site ID NO 848306  759 774 GCAACTTCAAGGCCAG 84 N/A N/A 1236 848217  149  164 AGCCAGTCTCACTGCC30  3624  3639 1237 848233  362  377 GCTGACGGTGCCCATG 51  3837  38521238 848249  487  502 GAACGCAAGGCTAGCA  5  3962  3977 1239 848790 N/AN/A CCAAATCGGAACCCAC 88  4478  4493 1240 848806 N/A N/A ACAGAACTTTCCCTTC57  5114  5129 1241 848822 N/A N/A TAAATTCGATTCCCAC 32  5600  5615 1242848838 N/A N/A AGGCAGTAATGGGCAA 92  6461  6476 1243 848854 N/A N/ATCCAACACTGAGGACC 93  6811  6826 1244 848870 N/A N/A AAAGGCAACACTTCTT 61 7222  7237 1245 848886 N/A N/A CAATATTTACTGGTTG 57  7409  7424 1246848902 N/A N/A CTTACAAATTACAACA 59  7751  7766 1247 848264  622  637TCTGACTGCGAGAGGT 71  7895  7910 1248 466849  675  690 TGGTGAGGTATCCCCG48  7948  7963 1249 468444  691  706 AAGACATGCAGGATCT 30  7964  79791250 848918 N/A N/A GACCAATGGGTTTGAT 13  8311  8326 1251 848934 N/A N/AGAGAATACCCAGTCCC 50  8550  8565 1252 848950 N/A N/A GCTCAAGGGAAAGGCC  3 9044  9059 1253 848966 N/A N/A GGACAAGAGTGCATCA  6 10011 10026 1254848982 N/A N/A GGCCTTACCTGATCAA 12 10437 10452 1255 848998 N/A N/ACTAGAACCCTTCATTC  0 11304 11319 1256 849014 N/A N/A TCTCACCTGGTTGGAA 3111856 11871 1257 849030 N/A N/A GTTAAGAGTGCAGGGT 90 12708 12723 1258849044 N/A N/A GGATACACAGGCTCGC 92 13108 13123  337 849046 N/A N/ACCGTAGGTCTTGGCTA 65 13166 13181 1259 849062 N/A N/A AGCTGTACCTGGGTTC 7913888 13903 1260 849078 N/A N/A CCTAGAGGAACCACTA 51 14340 14355 1261849094 N/A N/A ACATGAATTTCAGGCA 73 14731 14746 1262 849110 N/A N/ATCTCAGCCAGGCCAAA  0 15411 15426 1263 849126 N/A N/A CCCTATTATAGCCTTT 6715920 15935 1264 848321  890  905 CTCCACCAGGCTGCCT  0 16282 16297 1265848336  986 1001 CCCGTCCTCCTCGGGC  0 16378 16393 1266 849142 N/A N/ACCATCAGACGGCCGTG 48 16417 16432 1267 848350 1020 1035 TGTCACACTTGCTGGC45 16650 16665 1268 848365 1064 1079 GGCATCCCGGCCGCTG 62 16694 167091269 468460 1075 1090 TTGGCCACGCCGGCAT  0 16705 16720   48 848381 10981113 GCAGGCTGCGCATGCT 42 16728 16743 1270 849158 N/A N/ATGCAAGCAGAAGATAG 39 17153 17168 1271 849174 N/A N/A CAGGAACTGACCTGAC 2117808 17823 1272 849190 N/A N/A TACAGCTGCTAGTTAT 59 18086 18101 127318399 18414 849206 N/A N/A GTGCAGGCCATGGTCT 58 19054 19069 1274 849222N/A N/A ATATACGAACTCAGGG  0 19426 19441 1275 849238 N/A N/AGGCCTTAAGAGAACAG 46 19734 19749 1276 848397 1178 1193 GACCAGCTGGCTTTTC 2 20009 20024 1277 849254 N/A N/A CCCAAAGAGTATTGGG  0 20552 20567 1278849270 N/A N/A AACTATTCGGTGTATC 67 21051 21066 1279 848413 1358 1373CCCAACTGTGATGACC  0 21330 21345 1280 848429 1485 1500 CAAAGCAGGTGCTGCA36 21457 21472 1281 849286 N/A N/A AGGTAAGACAGCCTCC 53 21856 21871 1282848445 1619 1634 GATGACATCTTTGGCA 59 22112 22127 1283 848461 1732 1747GACCATACAGTCCTGC 17 22514 22529 1284 848477 1816 1831 GAGAAACTGGAGCAGC 0 22598 22613 1285 849302 N/A N/A TAGCAGATCGCTGACC 67 22948 22963 1286849318 N/A N/A ACTCAAGCACCCTCAT  0 23394 23409 1287 848491 1985 2000CATGCTGGCCTCAGCT 11 23605 23620 1288 848507 2008 2023 TGGTGGCAGTGGACAC71 23628 23643 1289 849334 N/A N/A AATGAGGCAGGTAATA 19 23879 23894 1290849350 N/A N/A CCTCAGGGCACCATCC 72 24293 24308 1291 849366 N/A N/AGGGAAAACACCATCTT 31 25120 25135 1292 849382 N/A N/A AGCATCTACCTGGCAA 3925666 25681 1293 849398 N/A N/A GTGTACCCAGGGCAAG 59 26373 26388 1294849414 N/A N/A AAGCACATGTCTAGCG  8 27331 27346 1295 848523 2246 2261AGTCAGGGTCCAGCCC 63 27389 27404 1296 848539 2487 2502 CATTTTAAAGCTCAGC87 27630 27645 1297 848555 2580 2595 CTCAAGGGCCAGGCCA 70 27723 277381298 848571 2708 2723 GCACCCGAGCACAGCT 75 27851 27866 1299 848586 27432758 GCCCACGGACATCGGC 58 27886 27901 1300 848602 2778 2793CTGGCACGGAACAAGA  8 27921 27936 1301 848618 2904 2919 GATGAGGGCCATCAGC87 28047 28062 1302 848633 2978 2993 GGCTAGATGCCATCCA 64 28121 281361303 848648 3093 3108 CCGCAGGCCACCTTTG 12 28236 28251 1304 848664 31463161 AGACAGTGCATGCACC 10 28289 28304 1305 848680 3222 3237TCTGGTTCCAGGTTTC 89 28365 28380 1306 848695 3255 3270 TTCCTGCTGTGTGAGC63 28398 28413 1307 848711 3304 3319 AAGAAGAGGCTTGGCT 38 28447 284621308 848727 3382 3397 CACCGAGCTTCCTGGT 66 28525 28540 1309 848743 34463461 GTGAATCAGGCCTGGG 31 28589 28604 1310 848758 3558 3573GGACAGACCCAAAAGA 71 28701 28716 1311 848774 3641 3656 TGCTACAAAACCCAGA95 28784 28799 1312

TABLE 18Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ ID: 1 SEQ ID: 1 % SEQ ID: 2 SEQ ID: 2 SEQ ISIS No Start SiteStop Site Sequence inhibition Start Site Stop Site ID NO 848218  153 168 CCCGAGCCAGTCTCAC  1  3628  3643 1313 848234  368  383CCTGGAGCTGACGGTG 18  3843  3858 1314 848250  488  503 GGAACGCAAGGCTAGC77  3963  3978 1315 848791 N/A N/A TTCCAAACCAAATCGG 46  4485  4500 1316848807 N/A N/A GGACAAAACTGCAAGT 42  5170  5185 1317 848823 N/A N/ATTCTAAATTCGATTCC 61  5603  5618 1318 848839 N/A N/A TTAGAAAGGCAGTAAT 30 6467  6482 1319 848855 N/A N/A CAAGGACGGCACCAAG 37  6881  6896 1320848871 N/A N/A GAAGGGATGACTAAGT  0  7242  7257 1321 848887 N/A N/AAGGAAACCGTGGACCT 60  7437  7452 1322 848903 N/A N/A CCTACTTACAAATTAC  4 7755  7770 1323 848265  633  648 GGGCAGTGCGCTCTGA 78  7906  7921 1324848276  676  691 TTGGTGAGGTATCCCC 66  7949  7964 1325 848291  692  707GAAGACATGCAGGATC 61  7965  7980 1326 848919 N/A N/A GGCAGACCAATGGGTT 10 8315  8330 1327 848935 N/A N/A TAAGATTTGAAGCACT 60  8580  8595 1328848951 N/A N/A CTGAACTGTAAGCTCA 46  9055  9070 1329 848967 N/A N/AGGCACTAGCAGGAGTT 62 10031 10046 1330 848983 N/A N/A ATGGATTCAGCTCAGA 6610680 10695 1331 848999 N/A N/A TCCAACTAGAACCCTT 87 11309 11324 1332849015 N/A N/A CACTGAAGAGGTCTCA 17 11867 11882 1333 849031 N/A N/AAGGAACCTGAAGATCA 72 12756 12771 1334 849044 N/A N/A GGATACACAGGCTCGC 8513108 13123  337 849047 N/A N/A TAATAATGCCCCCGTA 49 13177 13192 1335849063 N/A N/A AGTTAGCCCAGGTGAG  6  1395 13966 1336 849079 N/A N/ACGCCACCCTAGAGGAA  0 14346 14361 1337 849095 N/A N/A AGCAATTCAGTGGCTG 4914759 14774 1338 849111 N/A N/A CTCCAGCGCAGGTCTC 35 15433 15448 1339849127 N/A N/A GTGTAAAATAAAGCCC 77 15934 15949 1340 848322  900  915GGAGATACACCTCCAC 15 16292 16307 1341 848337  987 1002 TCCCGTCCTCCTCGGG 8 16379 16394 1342 849143 N/A N/A GGACAGTGACAGCTGG 39 16524 16539 1343848351 1021 1036 CTGTCACACTTGCTGG  8 16651 16666 1344 848366 1070 1085CACGCCGGCATCCCGG  3 16700 16715 1345 468460 1075 1090 TTGGCCACGCCGGCAT 0 16705 16720   48 848382 1100 1115 GCGCAGGCTGCGCATG  0 16730 167451346 849159 N/A N/A ATGCAAGTGAGTGAGT 74 17187 17202 1347 849175 N/A N/AGGAAAGAACAGCCTCC 44 17842 17857 1348 849191 N/A N/A GATACAGACACCCACC 3718250 18265 1349 849207 N/A N/A TACCATAACTCCCCAC 37 19123 19138 1350849223 N/A N/A ATCAAACTAGCCAACC  0 19482 19497 1351 849239 N/A N/AGAAGACGGAGTAAGGC 81 19795 19810 1352 848398 1183 1198 GGCTGGACCAGCTGGC 0 20014 20029 1353 849255 N/A N/A GGCCCAAAGAGTATTG  0 20554 20569 1354849271 N/A N/A GAAACTATTCGGTGTA 38 21053 21068 1355 848414 1370 1385GGCATTGGTGGCCCCA 19 21342 21357 1356 848430 1492 1507 TGTGACACAAAGCAGG84 21464 21479 1357 849287 N/A N/A CGAAGGTAAGCCGCCT 56 21889 21904 1358848446 1626 1641 CCTCATTGATGACATC  0 22119 22134 1359 848462 1733 1748TGACCATACAGTCCTG 19 22515 22530 1360 848478 1842 1857 GCTCGCCCCGCCGCTT35 22624 22639 1361 849303 N/A N/A GGCTGAGTCCAGAGTA 89 22962 22977 1362849319 N/A N/A GAAATGGGAATCTGCT  0 23454 23469 1363 848492 1986 2001CCATGCTGGCCTCAGC 32 23606 23621 1364 849335 N/A N/A CAGCTATAGAGCGGCA  024045 24060 1365 849351 N/A N/A GAACAGCTCAGCCTCA  0 24304 24319 1366849367 N/A N/A AGCCTTAGTTTCTCAG 25 25135 25150 1367 848508 2047 2062TCCCAGTGGGAGCTGC  0 25378 25393 1368 849383 N/A N/A CCACAGCATCTACCTG 8025670 25685 1369 849399 N/A N/A CCAGGAAGAGCACCTG 17 26406 26421 1370849415 N/A N/A AGAAAGCACATGTCTA  0 27334 27349 1371 848524 2250 2265AGCCAGTCAGGGTCCA 55 27393 27408 1372 848540 2490 2505 AACCATTTTAAAGCTC45 27633 27648 1373 848556 2582 2597 CACTCAAGGGCCAGGC 37 27725 277401374 848572 2709 2724 AGCACCCGAGCACAGC 89 27852 27867 1375 848587 27522767 AGTCATTCTGCCCACG 78 27895 27910 1376 848603 2779 2794CCTGGCACGGAACAAG 51 27922 27937 1377 848619 2906 2921 GAGATGAGGGCCATCA80 28049 28064 1378 848634 2999 3014 GCGCACCTGTCTCCAG  0 28142 281571379 848649 3110 3125 TCCTAGGTGATGGCTC 80 28253 28268 1380 848665 31483163 TGAGACAGTGCATGCA 37 28291 28306 1381 848681 3223 3238CTCTGGTTCCAGGTTT 43 28366 28381 1382 848696 3256 3271 GTTCCTGCTGTGTGAG78 28399 28414 1383 848712 3306 3321 GTAAGAAGAGGCTTGG 75 28449 284641384 848728 3383 3398 TCACCGAGCTTCCTGG 50 28526 28541 1385 848744 34513466 GGCCAGTGAATCAGGC 17 28594 28609 1386 848759 3563 3578AGAGAGGACAGACCCA 83 28706 28721 1387 848775 3644 3659 AAATGCTACAAAACCC67 28787 28802 1388 848307  760  775 GGCAACTTCAAGGCCA  0 N/A N/A 1389

TABLE 19Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ ID: 1 SEQ ID: 1 % SEQ ID: 2 SEQ ID: 2 SEQ ISIS No Start SiteStop Site Sequence inhibition Start Site Stop Site ID NO 848219  181 196 CGCTGCTGCAACGACG 66  3656  3671 1390 848235  378  393ACCAGGACCGCCTGGA  0  3853  3868 1391 848251  489  504 CGGAACGCAAGGCTAG62  3964  3979 1392 848792 N/A N/A GTCATAAAGAAATTGC 47  4593  4608 1393848808 N/A N/A TGGCAGAATTTTCCCC 17  5249  5264 1394 848824 N/A N/ATTTCATTCTAAATTCG 58  5608  5623 1395 848840 N/A N/A CTTTACCCAAAGCCTT  0 6523  6538 1396 848856 N/A N/A CATAGCGGGAGAACTT 51  6908  6923 1397848872 N/A N/A GCAGAAGGGATGACTA 53  7245  7260 1398 848888 N/A N/AAGCCAGGAAACCGTGG 63  7441  7456 1399 848904 N/A N/A CCCTACTTACAAATTA 40 7756  7771 1400 848266  640  655 AGGCGGCGGGCAGTGC 31  7913  7928 1401848277  677  692 CTTGGTGAGGTATCCC 65  7950  7965 1402 848292  693  708GGAAGACATGCAGGAT 64  7966  7981 1403 848920 N/A N/A AGGCAGACCAATGGGT 63 8316  8331 1404 848936 N/A N/A CTAGAAGGTGGTGCAG  0  8619  8634 1405848952 N/A N/A CCATAGTCAACTGTAC  7  9097  9112 1406 848968 N/A N/AAGCCAATGGGAGGCAC 15 10042 10057 1407 848308  761  776 GGGCAACTTCAAGGCC 7 10522 10537 1408 848984 N/A N/A GGGCAGAGCAAATGGA  0 10691 10706 1409849000 N/A N/A GTTCACCCCAAGCTCT 29 11420 11435 1410 849016 N/A N/ACTCCACTGAAGAGGTC 66 11870 11885 1411 849032 N/A N/A GATTTAACCCTCCAAA  012814 12829 1412 849044 N/A N/A GGATACACAGGCTCGC 86 13108 13123  337849048 N/A N/A GTTAATAATGCCCCCG 80 13179 13194 1413 849064 N/A N/AGCTGAGTTAGCCCAGG 51 13955 13970 1414 849080 N/A N/A AAACAGTGCTCGCCAC 2914356 14371 1415 849096 N/A N/A TACAGAGCAATTCAGT 33 14764 14779 1416849112 N/A N/A TGCCAGGCAGGTCCAG  0 15506 15521 1417 849128 N/A N/ACAATATCTAACAATAA  0 15977 15992 1418 848323  905  920 GTCTAGGAGATACACC72 16297 16312 1419 848338  988 1003 GTCCCGTCCTCCTCGG 33 16380 163951420 849144 N/A N/A AATCAGCAGGTGGCTG  0 16579 16594 1421 848352 10221037 ACTGTCACACTTGCTG 45 16652 16667 1422 848367 1072 1087GCCACGCCGGCATCCC 27 16702 16717 1423 468460 1075 1090 TTGGCCACGCCGGCAT 2 16705 16720   48 848383 1105 1120 AGCACGCGCAGGCTGC  0 16735 167501424 849160 N/A N/A CCACATCTGCCTGGCC  0 17226 17241 1425 849176 N/A N/ATGCCAGGTCATGCAAT  0 17954 17969 1426 849192 N/A N/A GGGATACAGACACCCA 4818252 18267 1427 849208 N/A N/A CCATACCATAACTCCC 36 19126 19141 1428849224 N/A N/A CAATTACACCAGCAAT 64 19496 19511 1429 849240 N/A N/ACTGCGAAAATATTTTT 32 19835 19850 1430 848399 1188 1203 CCACAGGCTGGACCAG 8 20019 20034 1431 849256 N/A N/A CCTCAACCGCTCCCAT 15 20631 20646 1432849272 N/A N/A GAGATAGGAAACTATT 19 21060 21075 1433 848415 1388 1403GGTCACCGGCTGGTCT  0 21360 21375 1434 848431 1500 1515 TCCCACTCTGTGACAC93 21472 21487 1435 849288 N/A N/A ACAGACACTAAGCTCT 76 21910 21925 1436848447 1628 1643 GGCCTCATTGATGACA  0 22121 22136 1437 848463 1734 1749CTGACCATACAGTCCT 15 22516 22531 1438 848479 1850 1865 CTCCATGCGCTCGCCC39 22632 22647 1439 849304 N/A N/A CTAGAGATGGCTGAGT 56 22970 22985 1440849320 N/A N/A ACGGAAATGGGAATCT  0 23457 23472 1441 848493 1990 2005GTCCCCATGCTGGCCT 33 23610 23625 1442 849336 N/A N/A ATACAGAGATGTTAAG 3024079 24094 1443 849352 N/A N/A TCTTATTCCAGGCTGG 23 24362 24377 1444849368 N/A N/A AGCTGACCAGCTGTGT  0 25180 25195 1445 848509 2064 2079TGCCAAGGTCCTCCAC 18 25395 25410 1446 849384 N/A N/A CAAAAACAGACCCAGC 1125733 25748 1447 849400 N/A N/A AAATAGATGCTCCAGG 35 26465 26480 1448849416 N/A N/A CGAGGAAAAGAAAGCA 20 27342 27357 1449 848525 2254 2269CTGCAGCCAGTCAGGG  1 27397 27412 1450 848541 2494 2509 TCGGAACCATTTTAAA 0 27637 27652 1451 848557 2585 2600 CCCCACTCAAGGGCCA  0 27728 277431452 848573 2710 2725 CAGCACCCGAGCACAG 23 27853 27868 1453 848588 27552770 AAAAGTCATTCTGCCC 62 27898 27913 1454 848604 2780 2795GCCTGGCACGGAACAA 55 27923 27938 1455 848620 2909 2924 CTGGAGATGAGGGCCA24 28052 28067 1456 848635 3021 3036 GCACAGCCTGTGACCA 71 28164 281791457 848650 3120 3135 GCCGAGTCAGTCCTAG 23 28263 28278 1458 848666 31503165 GCTGAGACAGTGCATG 47 28293 28308 1459 468497 3224 3239CCTCTGGTTCCAGGTT 77 28367 28382 1460 848697 3257 3272 AGTTCCTGCTGTGTGA44 28400 28415 1461 848713 3308 3323 AAGTAAGAAGAGGCTT  0 28451 284661462 848729 3384 3399 CTCACCGAGCTTCCTG 76 28527 28542 1463 848745 34563471 CGCCAGGCCAGTGAAT 44 28599 28614 1464 848760 3565 3580ACAGAGAGGACAGACC 45 28708 28723 1465 848776 3661 3676 AGTCACCATATTAATA63 28804 28819 1466

TABLE 20Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2SEQ ID: 1 SEQ ID: 1 % SEQ ID: 2 SEQ ID: 2 SEQ ISIS No Start SiteStop Site Sequence inhibition Start Site Stop Site ID NO 848432 15351550 TGCAATGCCAGCCACG  0 N/A N/A 1467 848220  184  199 AGCCGCTGCTGCAACG57  3659  3674 1468 848236  381  396 GCCACCAGGACCGCCT 28  3856  38711469 848252  492  507 CCTCGGAACGCAAGGC 38  3967  3982 1470 848793 N/AN/A AGCATGAGTTCTGTGT 43  4607  4622 1471 848809 N/A N/A CAGCACACTCAGACAG47  5288  5303 1472 848825 N/A N/A AAAAGGATTGGTCTAA  0  5652  5667 1473848841 N/A N/A CCCTTTACCCAAAGCC 71  6525  6540 1474 848857 N/A N/AAATCAGCCTTCAAGGG 39  6928  6943 1475 848873 N/A N/A GGCAGAAGGGATGACT 35 7246  7261 1476 848889 N/A N/A TCGACAACAGGTTTTC 58  7576  7591 1477848905 N/A N/A TGACATGGAAGAAACC 34  7801  7816 1478 848267  642  657GCAGGCGGCGGGCAGT  0  7915  7930 1479 848278  678  693 TCTTGGTGAGGTATCC52  7951  7966 1480 848293  694  709 TGGAAGACATGCAGGA 26  7967  79821481 848921 N/A N/A TGCATTGGCACAAGAA 44  8340  8355 1482 848937 N/A N/AAGTTAGAGGCCAGGAA 25  8645  8660 1483 848953 N/A N/A TCCCATAGTCAACTGT 11 9099  9114 1484 848969 N/A N/A TTGCAAAGCTTCCAGT  7 10065 10080 1485468446  775  790 ATGTAGTCGACATGGG 38 10536 10551 1486 848985 N/A N/ACGAGAAGTGGAAACCA  0 10727 10742 1487 849001 N/A N/A GTGAAAGCTGAGTTCA  611431 11446 1488 849017 N/A N/A GGGTGGTAATTTGTCA 15 12045 12060 1489849033 N/A N/A AATAACTGATTTAACC 12 12821 12836 1490 849044 N/A N/AGGATACACAGGCTCGC 82 13108 13123  337 849049 N/A N/A GTTCATTCCACTGCTT 3013216 13231 1491 849065 N/A N/A CAACGCACATCGAGCA 39 14038 14053 1492849081 N/A N/A ACCAAACAGTGCTCGC 48 14359 14374 1493 849097 N/A N/AAATAAGGTCTGGCTCA 43 14829 14844 1494 849113 N/A N/A ACAGATGCCAGGCAGG 1015511 15526 1495 849129 N/A N/A TCAATATCTAACAATA  0 15978 15993 1496848324  917  932 CTGTATGCTGGTGTCT  0 16309 16324 1497 848339  989 1004GGTCCCGTCCTCCTCG  0 16381 16396 1498 849145 N/A N/A CATGAGAAAGACCCCC  016611 16626 1499 848353 1024 1039 TGACTGTCACACTTGC 56 16654 16669 1500848368 1073 1088 GGCCACGCCGGCATCC  0 16703 16718 1501 468460 1075 1090TTGGCCACGCCGGCAT  0 16705 16720   48 848384 1109 1124 GTTGAGCACGCGCAGG58 16739 16754 1502 849161 N/A N/A CTCCACCACATCTGCC 30 17231 17246 1503849177 N/A N/A CGTGCCAGGTCATGCA 74 17956 17971 1504 849193 N/A N/ATGGGATACAGACACCC  5 18253 18268 1505 849209 N/A N/A TAAAAGACTCCATGCC  019151 19166 1506 849225 N/A N/A GCAATTACACCAGCAA 84 19497 19512 1507849241 N/A N/A GCACAGAGTGATGGTT 66 19908 19923 1508 848400 1190 1205CCCCACAGGCTGGACC 34 20021 20036 1509 849257 N/A N/A CTGCACCGGGCATGCG  020686 20701 1510 849273 N/A N/A CCCTACCATAGCCAGG 36 21118 21133 1511848416 1394 1409 CCCCAGGGTCACCGGC 38 21366 21381 1512 849289 N/A N/AGCACACAGACACTAAG  0 21914 21929 1513 848448 1633 1648 AACCAGGCCTCATTGA 0 22126 22141 1514 848464 1735 1750 GCTGACCATACAGTCC  9 22517 225321515 849305 N/A N/A CTATCCTGTAGCATCA 73 23060 23075 1516 849321 N/A N/AGACGGAAATGGGAATC  0 23458 23473 1517 848480 1875 1890 GGCAGACCAGCTTGCC 0 23495 23510 1518 848494 1991 2006 GGTCCCCATGCTGGCC  0 23611 236261519 849337 N/A N/A TAGCACTCATCATTTC 33 24098 24113 1520 849353 N/A N/AGACGAGAATCAACTCT  6 24383 24398 1521 849369 N/A N/A CACCAGGACTCCTGTG  425209 25224 1522 848510 2074 2089 GGCTTGTGGGTGCCAA 15 25405 25420 1523849385 N/A N/A CTCCAAGTGGAGTGGG  0 25786 25801 1524 849401 N/A N/AACTAAAATAGATGCTC 34 26469 26484 1525 849417 N/A N/A TGCCAGAGCCCGAGGA  027352 27367 1526 848526 2259 2274 GGGCACTGCAGCCAGT  0 27402 27417 1527848542 2500 2515 GACAAGTCGGAACCAT 77 27643 27658 1528 848558 2598 2613GGCAAGGAGGCTGCCC  0 27741 27756 1529 848574 2711 2726 GCAGCACCCGAGCACA70 27854 27869 1530 848589 2756 2771 TAAAAGTCATTCTGCC 56 27899 279141531 848605 2781 2796 TGCCTGGCACGGAACA 44 27924 27939 1532 848621 29152930 AGTTAGCTGGAGATGA  0 28058 28073 1533 848636 3027 3042ACCAAGGCACAGCCTG 70 28170 28185 1534 848651 3121 3136 TGCCGAGTCAGTCCTA69 28264 28279 1535 848667 3163 3178 GTGGAGCGGGTTGGCT 26 28306 283211536 848682 3225 3240 CCCTCTGGTTCCAGGT 23 28368 28383 1537 848698 32593274 TCAGTTCCTGCTGTGT 30 28402 28417 1538 848714 3311 3326GTGAAGTAAGAAGAGG 47 28454 28469 1539 848730 3385 3400 ACTCACCGAGCTTCCT63 28528 28543 1540 848746 3469 3484 TTAGAAGCATCTCCGC 44 28612 286271541 848761 3569 3584 GGCAACAGAGAGGACA 79 28712 28727 1542 848777 36663681 TAAAAAGTCACCATAT  9 28809 28824 1543

Example 2: Dose-Dependent Antisense Inhibition of Human PCSK9 in HepG2Cells by 3-10-3 cEt Gapmers

Select gapmers from Example 1 exhibiting in vitro inhibition of PCSK9mRNA were tested at various doses in HepG2 cells. The antisenseoligonucleotides were tested in a series of experiments that had similarculture conditions. The results for each experiment are presented inseparate tables shown below.

Study 1

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 46.88 nM, 187.5 nM, 750 nM, and 3,000 nMconcentrations of antisense oligonucleotide, as specified in the Tablesbelow. ISIS 405879, previously disclosed in WO2008066776 was alsoincluded in the study as a benchmark oligonucleotide. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andPCSK9 mRNA levels were measured by quantitative real-time PCR. HumanPCSK9 primer probe set from ABI (ID4 Hs03037355_m1) was used to measuremRNA levels. PCSK9 mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of PCSK9, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PCSK9 mRNA levels were reduced in a dose-dependentmanner in antisense oligonucleotide treated cells. Several of the newlydesigned antisense oligonucleotides demonstrated superior efficacycompared to the previously disclosed oligonucleotide, ISIS 405879.Specifically, ISIS 848542, ISIS 848593, ISIS 848597, ISIS 848598, ISIS848630, ISIS 848833, ISIS 849040, ISIS 849171, and ISIS 849236demonstrated superior efficacy compared to the previously disclosedoligonucleotide, ISIS 405879.

TABLE 21 ISIS 46.875 187.5 750 3,000 IC₅₀ No nM nM nM nM (μM) 405879 136 3 29 >3.00 466847 1 19 65 94 0.47 468497 12 27 60 83 0.48 848219 0 054 88 0.91 848241 0 5 31 63 1.78 848283 18 27 54 85 0.48 848306 0 19 5391 0.62 848323 0 0 0 50 >3.00 848376 0 3 12 43 >3.00 848430 0 30 52 810.62 848431 67 79 92 96 <0.05 848500 6 30 55 82 0.54 848539 0 0 055 >3.00 848542 10 30 66 86 0.41 848560 12 32 76 91 0.33 848567 0 1 4289 0.85 848570 23 27 67 87 0.35 848572 18 44 71 81 0.30 848574 0 5 68 950.55 848575 0 17 51 80 0.75 848576 11 18 45 74 0.86 848583 3 18 62 880.52 848584 0 22 54 89 0.57 848585 1 22 67 92 0.45 848593 0 25 53 830.63 848596 18 40 76 93 0.26 848597 22 42 78 87 0.24 848598 0 18 62 950.53 848616 0 14 55 88 0.66 848618 0 0 56 77 0.96 848619 3 7 41 74 1.07848629 21 34 78 78 0.31 848630 7 22 65 83 0.50 848632 25 34 68 89 0.29848635 0 0 49 83 0.92 848636 5 34 77 76 0.42 848649 5 10 43 78 0.89848651 4 0 48 88 0.88 848652 0 6 28 80 1.16 848656 1 13 52 80 0.73848670 0 38 84 85 0.39 848671 26 28 70 81 0.34 848672 0 11 27 73 1.37848674 36 31 69 89 0.24 848677 2 32 69 90 0.41 848679 0 11 55 81 0.73848680 11 20 64 81 0.52 848685 0 14 33 62 1.63 848687 0 25 63 90 0.51848688 0 23 61 89 0.55 848690 15 21 78 84 0.39 848692 31 45 80 97 0.17848693 8 23 69 88 0.44 848694 27 43 75 93 0.21 848701 0 10 39 77 1.02848704 11 5 43 78 0.94 848725 0 9 51 81 0.79 848729 26 33 63 92 0.30848731 0 15 60 89 0.55 848732 12 71 67 90 0.21 848736 6 18 40 73 1.00848740 2 18 59 86 0.57 848748 9 42 60 85 0.39 848756 3 1 55 85 0.72848759 18 37 75 94 0.28 848761 20 35 68 81 0.35 848764 0 8 37 64 1.52848770 0 4 63 90 0.63 848771 3 23 57 93 0.50 848772 15 32 66 82 0.40848774 0 8 64 90 0.61 848790 0 0 33 73 1.49 848795 9 42 62 91 0.35848811 7 5 46 86 0.75 848814 17 20 64 89 0.43 848826 7 57 77 93 0.24848830 0 35 45 86 0.62 848832 0 40 71 78 0.52 848833 19 43 68 81 0.32848838 0 36 72 89 0.39 848841 5 24 62 86 0.50 848844 0 22 61 89 0.53848849 9 48 79 92 0.26 848850 0 38 68 97 0.43 848854 2 7 58 84 0.66848875 3 17 58 79 0.65 848890 0 6 27 65 1.74 848898 7 14 79 87 0.43848927 12 25 75 95 0.35 848928 0 0 45 78 1.01 848971 0 21 54 82 0.65848999 6 29 34 64 1.38 849007 0 4 41 78 1.02 849008 0 39 74 90 0.42849013 4 17 47 86 0.67 849016 0 23 56 79 0.65 849020 0 13 40 67 1.28849021 10 34 65 80 0.44 849030 2 28 58 86 0.52 849036 2 1 37 76 1.16849040 28 55 79 96 0.15 849044 26 39 72 91 0.28 849048 0 30 60 79 0.61849069 12 12 40 74 1.02 849071 2 31 65 92 0.42 849084 0 0 0 43 >3.00849085 20 39 64 89 0.31 849086 13 40 69 81 0.36 849106 20 24 55 80 0.53849120 0 15 61 80 0.64 849123 9 48 82 84 0.27 849133 0 0 29 70 1.62849135 25 54 79 95 0.17 849162 0 4 35 75 1.19 849169 34 63 72 83 0.12849171 9 60 63 83 0.30 849177 0 16 69 75 0.68 849178 0 22 53 83 0.65849181 6 8 39 84 0.86 849189 24 44 81 90 0.21 849200 0 0 34 70 1.48849205 7 29 51 88 0.52 849216 0 15 28 66 1.66 849225 23 47 78 94 0.21849227 7 35 74 94 0.34 849233 9 42 68 93 0.32 849236 0 1 57 88 0.73849239 18 38 74 86 0.30 849266 0 22 66 86 0.51 849267 18 69 72 94 0.17849285 3 18 45 84 0.71 849288 0 0 0 52 >3.00 849303 18 33 57 72 0.54849305 25 28 62 82 0.40 849309 10 18 59 82 0.57 849331 0 10 65 93 0.57849332 0 0 48 76 1.09 849345 0 0 62 80 0.78 849354 0 0 43 64 1.49 8493575 21 45 75 0.84 849358 0 10 45 79 0.88 849359 3 22 46 88 0.62 849372 015 29 82 1.04 849383 2 0 33 71 1.43 849386 11 18 51 84 0.60

Study 2

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 78.13 nM, 312.5 nM, 1,250 nM, and 5,000 nMconcentrations of antisense oligonucleotide, as specified in the Tablesbelow. ISIS 431131, previously disclosed in WO2014179620, was alsoincluded in the study as a benchmark oligonucleotide. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andPCSK9 mRNA levels were measured by quantitative real-time PCR. HumanPCSK9 primer probe set from ABI (ID #Hs03037355_m1) was used to measuremRNA levels. PCSK9 mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of PCSK9, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PCSK9 mRNA levels were reduced in a dose-dependentmanner in antisense oligonucleotide treated cells. Several of the newlydesigned antisense oligonucleotides demonstrated superior efficacycompared to the previously disclosed oligonucleotide, ISIS 431131.

TABLE 22 ISIS 78.125 312.5 1,250 5,000 IC₅₀ No nM nM nM nM (μM) 43113118 16 30 55 >5 849169 23 57 86 92 0.28 859341 24 48 69 85 1.54 859346 2754 80 94 0.27 859358 0 30 69 89 0.76 859361 28 53 73 94 0.30 859363 3850 79 82 0.20 859384 19 59 76 94 0.30 859387 36 66 79 96 0.16 859392 1433 63 81 0.96 859393 20 59 90 96 0.27 859394 43 65 80 96 0.12 859395 3751 82 91 0.21 859401 27 64 88 94 0.20 859408 0 59 61 92 0.55 859419 2258 77 84 0.31 859459 33 55 91 93 0.19 859462 31 56 78 96 0.24 859469 2648 76 83 0.36 859472 19 35 67 87 0.56 859504 19 47 59 80 0.57 859529 1950 88 94 0.32 859531 33 52 76 69 0.29 859532 24 55 79 90 0.30 859533 940 77 91 0.51 859534 25 49 85 85 0.70 859551 25 41 51 78 0.69 859552 017 53 78 1.36 859553 24 45 79 94 0.35 859561 28 54 71 92 0.30 859571 3534 76 73 0.43 859584 19 51 85 95 0.32 859598 33 55 73 83 0.25 859601 2339 63 90 0.50 859603 0 6 70 71 1.25 859605 9 42 67 83 0.62 859611 21 2247 76 1.16

Example 3: Tolerability of 3′-Conjugated 3-10-3 cEt Gapmers TargetingHuman PCSK9 in BALB/c Mice

ISIS oligonucleotides selected from the studies above were conjugatedwith 3′-THA-C6-GalNAc3-(3R,5S)-5-(hydroxymethyl) pyrrolidin-3-olphosphate endcap (henceforth referred to as 3′-THA). The hundred andsixty two 3′-THA-conjugated ISIS antisense oligonucleotides evaluatedfor changes in the levels of plasma chemistry markers are presented inthe Table below. ‘Parent Oligo’ indicates the ISIS oligonucleotide thathas been described in the studies above and that was conjugated with3′-THA and tested in this study.

TABLE 23 3′-conjugated 3-10-3 cEt gapmers selected for tolerabilityevaluation in BALB/c mice ISIS Parent SEQ ID No Oligo NO 863413 4668471096 863415 848219 1390 863416 848241 777 863418 848306 1236 863419848323 1419 863421 848430 1357 863422 848431 1435 863423 848500 905863424 848539 1297 863425 848542 1528 863426 848560 607 863427 8485671145 863428 848570 451 863429 848572 1375 863433 848583 1223 863434848584 377 863435 848585 452 863436 848593 763 863437 848596 994 863438848597 1071 863439 848598 1147 863440 848616 379 863441 848618 1302863442 848619 1378 863443 848629 1073 863444 848630 1149 863445 848632455 863446 848635 1457 863447 848636 1534 863448 848649 1380 863449848651 1535 863451 848656 844 863452 848670 691 863453 848671 768 863457848679 458 863458 848680 1306 863464 848693 384 863465 848694 459 863468848725 386 863472 848736 926 863473 848740 1233 863474 848748 619 863475848756 388 863476 848759 1387 863477 848761 1542 863478 848764 697863479 848770 1158 863480 848771 1235 863481 848772 389 863482 8487741312 863483 848790 1240 863484 848795 549 863485 848811 550 863486848814 781 863487 848826 474 863488 848830 782 863489 848832 939 863490848833 1016 863491 848838 1243 863492 848841 1474 863493 848844 629863494 848849 1017 863495 848850 1092 863496 848854 1244 863497 848875554 863498 848890 478 863499 848898 1094 863500 848927 871 863501 848928947 863502 848971 563 863503 848999 1332 863504 849007 877 863505 849008953 863506 849013 410 863507 849016 1411 863508 849020 644 863509 849021721 863510 849030 1258 863511 849036 645 863512 849040 955 863513 849044337 863514 849048 1413 863515 849069 724 863516 849071 881 863517 849084648 863518 849085 725 863519 849086 802 863520 849106 1111 863521 849120960 863522 849123 1188 863523 849133 728 863524 849135 885 863525 849162504 863526 849169 1043 863527 849171 1195 863531 849189 426 863532849200 968 863533 849205 427 863535 849225 1507 863536 849227 585 863537849233 1047 863538 849236 353 863539 849239 1352 863540 849266 1126863541 849267 1203 863542 849285 434 863543 849288 1436 863544 8493031362 863545 849305 1516 863546 849309 749 863547 849331 1213 863548849332 367 863549 849345 1061 863550 849354 523 863551 849357 754 863552849358 831 863553 849359 908 863554 849372 678 863555 849383 1369 863556849386 526 884269 859561 55 884270 859459 180 884271 859395 164 884272859603 218 884273 859504 284 884274 859394 87 884275 859598 141 884276859531 198 884277 859469 28 884278 859346 152 884279 859361 79 884280859472 276

BALB/c mice are a multipurpose mice model, frequently utilized forsafety and efficacy testing. The mice were administered a single dose ofoligonucleotide. Plasma levels of transaminases were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,NY). ISIS oligonucleotides that caused changes in the levels of ALTand/or AST outside the expected range for antisense oligonucleotideswere excluded in further studies. ISIS 863413, ISIS 863419, ISIS 863424,ISIS 863425, ISIS 863427, ISIS 863433, ISIS 863434, ISIS 863436, ISIS863437, ISIS 863438, ISIS 863439, ISIS 863441, ISIS 863444, ISIS 863445,ISIS 863448, ISIS 863452, ISIS 863472, ISIS 863473, ISIS 863474, ISIS863475, ISIS 863477, ISIS 863479, ISIS 863480, ISIS 863481, ISIS 863482,ISIS 863483, ISIS 863484, ISIS 863485, ISIS 863486, ISIS 863489, ISIS863490, ISIS 863491, ISIS 863493, ISIS 863494, ISIS 863495, ISIS 863496,ISIS 863497, ISIS 863498, ISIS 863499, ISIS 863502, ISIS 863506, ISIS863507, ISIS 863509, ISIS 863510, ISIS 863511, ISIS 863512, ISIS 863514,ISIS 863516, ISIS 863517, ISIS 863518, ISIS 863520, ISIS 863522, ISIS863524, ISIS 863525, ISIS 863526, ISIS 863527, ISIS 863531, ISIS 863533,ISIS 863536, ISIS 863537, ISIS 863538, ISIS 863539, ISIS 863541, ISIS863545, ISIS 863547, ISIS 863548, ISIS 863549, ISIS 863550, ISIS 863552,and ISIS 863553 were considered tolerable in this study and wereselected for further evaluation.

Example 4: Effect of Antisense Inhibition of PCSK9 in Transgenic MouseModel

A transgenic mouse model was developed at UCI using the human PCSK9genomic construct, which was contained in fosmid ABC7-611722G24 andrestricted with NheI to produce a DNA fragment containing the entiregenomic sequence as well as 8 Kb of 5′ and 0.4 Kb of 3′ noncodingsequence. PCSK9 transgenic mice were produced by random insertion viamicronucleus injection. Progeny expressed human PCSK9 mRNA in the liverand secreted human PCSK9 plasma protein.

The efficacy of 3′-THA-conjugated ISIS oligonucleotides was evaluated inthis model (referred to herein as Tg mice). The ISIS oligonucleotidestested are presented in the Table below. ‘Parent ISIS No’ indicates theISIS oligonucleotide that has been described in the studies above andthat was conjugated with 3′-THA and tested in this study.

TABLE 24 ISIS oligonucleotides tested in Tg mice Parent SEQ ISIS NoSequence ISIS No ID NO 863413 GAGGTATCCCCGGCGG 466847 1096 863419GTCTAGGAGATACACC 848323 1419 863424 CATTTTAAAGCTCAGC 848539 1297 863425GACAAGTCGGAACCAT 848542 1528 863427 TTGAATGGTGAAATGC 848567 1145 863433GGACATCGGCACATTG 848583 1223 863434 ACGGACATCGGCACAT 848584  377 863436AGCTCAATAAAAGTCA 848593  763 863437 CGGAACAAGAGCTCAA 848596  994 863438ACGGAACAAGAGCTCA 848597 1071 863439 CACGGAACAAGAGCTC 848598 1147 863441GATGAGGGCCATCAGC 848618 1302 863444 CAGAAAGCTAAGCCTC 848630 1149 863445CTAGATGCCATCCAGA 848632  455 863448 TCCTAGGTGATGGCTC 848649 1380 863452GCGAATGTGTACCCTG 848670  691 863472 GGAAAAAGTTCCATGC 848736  926 863473GTGATAACGGAAAAAG 848740 1233 863474 TGCCTTAGAAGCATCT 848748  619 863475AAAGATAAATGTCTGC 848756  388 863477 GGCAACAGAGAGGACA 848761 1542 863479AGCAAAACAGGTCTAG 848770 1158 863480 TTCAAGTTACAAAAGC 848771 1235 863481CCCAGAATAAATATCT 848772  389 863482 TGCTACAAAACCCAGA 848774 1312 863483CCAAATCGGAACCCAC 848790 1240 863484 CGAGAATACCTCCGCC 848795  549 863485GCTGAGTAAGGACTTG 848811  550 863486 AGAAAGTCAAAGGCTC 848814  781 863489TACATTTCAGACGGTG 848832  939 863490 AATAATCTCATGTCAG 848833 1016 863491AGGCAGTAATGGGCAA 848838 1243 863493 TCATGAATCAAGTCCA 848844  629 863494CAAATTATAGCAGCCA 848849 1017 863495 AACAAATTATAGCAGC 848850 1092 863496TCCAACACTGAGGACC 848854 1244 863497 CTACAAATGCAGGCAG 848875  554 863498CTCGACAACAGGTTTT 848890  478 863499 ACCAAATGCGGACCAA 848898 1094 863502GCAATTCGGTTTGTCC 848971  563 863506 GGAAAGGAACAGGCTC 849013  410 863507CTCCACTGAAGAGGTC 849016 1411 863509 GACAATGAAGAGGAGA 849021  721 863510GTTAAGAGTGCAGGGT 849030 1258 863511 ACAGAGAAATGCATGC 849036  645 863512GTTATTATTGAATGGT 849040  955 863514 GTTAATAATGCCCCCG 849048 1413 863516ACAACTGGATACATTG 849071  881 863517 CAATAGGCATCTACCA 849084  648 863518ACTCATCAATAGGCAT 849085  725 863520 CCTCAGGTGGAATCAG 849106 1111 863522GGAGAATAACAGTGAT 849123 1188 863524 ATAGACAAGGAAAGGG 849135  885 863525GTCTAGAAAAAGTCCT 849162  504 863526 AGGAAAGTCTCAGGGC 849169 1043 863527CTGTAGGAAAGTCTCA 849171 1195 863531 ACAGCTGCTAGTTATT 849189  426 863533TGCTACTGTCAACAGT 849205  427 863536 GGAAGATATTAGCAAT 849227  585 863537GCGGATTTCAGACTTG 849233 1047 863538 CAACATCAAATTCTGC 849236  353 863539GAAGACGGAGTAAGGC 849239 1352 863541 TTCTAAGTGCCACGGG 849267 1203 863545CTATCCTGTAGCATCA 849305 1516 863547 GGAGAAGTAAGGTCAC 849331 1213 863548AGGAGAAGTAAGGTCA 849332  367 863549 ATTTTAAAGCAACGGG 849345 1061 863550CCTACATGCCAGCCTG 849354  523 863552 TCTGAACATGGTAGGG 849358  831 863553GTAAGATGGAAAGAGA 849359  908

Treatment

PCSK9 transgenic mice were maintained on a 12-hour light/dark cycle andwere fed ad libitum normal Purina mouse chow. Animals were acclimatedfor at least 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in bufferedsaline (PBS) and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

The PCSK9 transgenic mice received subcutaneous injections of ISISoligonucleotide at a dose of 2.5 mg/kg twice per week for 2 weeks. Twogroups of mice received subcutaneous injections of PBS for 2 weeks. Thesaline-injected groups served as the control group to whicholigonucleotide-treated groups were compared.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction of these mice, plasma levels of transaminases (ALT and AST),cholesterol (CHOL), HDL cholesterol (HDL), LDL cholesterol (LDL), andtriglycerides (TRIG) were measured on day 12 using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of the liver or kidney function markersoutside the expected range for antisense oligonucleotides were excludedin further studies.

TABLE 25 Plasma chemistry markers in Tg mice plasma ALT AST CHOL HDL LDLTRIG Treatment (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) PBS 17 37114 73 35 65 PBS 23 102 105 68 32 76 863413 19 72 122 88 24 89 863419 1544 115 73 31 104 863424 18 34 197 112 79 105 863425 25 53 143 101 36 104863427 22 96 113 75 28 127 863433 17 54 92 67 19 61 863434 24 36 132 9835 102 863436 17 50 124 81 31 96 863437 21 69 126 91 30 81 863438 18 6657 39 15 54 863439 42 173 111 78 22 107 863441 20 57 84 58 20 56 86344419 41 149 101 42 116 863445 20 36 169 111 48 111 863448 24 44 107 69 2979 863452 20 67 114 78 23 100 863472 35 55 105 71 26 89 863473 23 67 152105 38 130 863474 21 115 118 78 32 45 863475 23 87 179 112 62 92 86347767 142 222 137 73 69 863479 29 60 142 96 32 90 863480 19 52 122 88 25 53863481 28 50 223 132 70 115 863482 20 43 168 105 5 46 129 863483 19 34174 110 47 139 863484 25 71 64 38 21 66 863485 19 71 170 113 42 110863486 18 40 117 78 28 121 863489 38 68 90 66 18 89 863490 19 41 99 7119 113 863491 20 40 148 99 35 146 863493 18 74 107 73 26 59 863494 19 41149 106 32 104 863495 17 34 136 95 32 116 863496 545 275 136 97 31 29863497 36 49 121 84 28 53 863498 23 44 181 122 42 98 863499 21 51 128102 18 58 863502 34 144 116 84 22 86 863506 30 44 188 119 51 108 86350719 46 108 78 18 100 863509 23 63 121 80 28 115 863510 40 71 149 111 2851 863511 31 61 101 70 23 83 863512 21 52 83 59 14 103 863514 22 65 9969 23 64 863516 18 52 117 73 33 122 863517 25 98 113 74 27 125 863518 35116 96 65 23 70 863520 21 102 107 68 31 76 863522 31 55 89 56 24 77863524 49 61 154 104 43 94 863525 20 58 208 116 66 155 863526 32 82 159114 37 59 863527 25 112 98 66 23 101 863531 835 454 268 149 67 84 8635330 109 276 152 80 98 863536 22 68 120 75 28 181 863537 29 102 134 97 2752 863538 16 42 126 76 39 89 863539 22 53 100 65 25 64 863541 19 41 9866 23 115 863545 28 53 109 73 25 114 863547 22 41 119 80 30 71 863548550 433 188 125 33 60 863549 17 36 109 70 33 72 863550 32 62 158 98 4377 863552 18 55 113 71 33 79 863553 18 43 103 62 30 86

Example 5: Tolerability of ISIS Oligonucleotides Targeting Human PCSK9in CD1 Mice

ISIS oligonucleotides selected from the studies above were conjugatedwith 5′-Trishexylamino-(THA)-C6GalNAC3 endcap (henceforth referred to as5′-THA). CD1® mice (Charles River, MA) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with ISIS antisense oligonucleotides, selected from studiesdescribed above and conjugated with 5′-THA, and evaluated for changes inthe levels of various plasma chemistry markers.

Study 1 Treatment

Groups of male CD1 mice were injected subcutaneously once a week for 6weeks with 15 mg/kg of ISIS oligonucleotides. One group of male CD1 micewas injected subcutaneously once a week for 6 weeks with PBS. Mice wereeuthanized 48 hours after the last dose, and organs and plasma wereharvested for further analysis.

The ISIS oligonucleotides tested are presented in the Table below.‘Unconjugated parent ISIS No.’ refers to the ISIS oligonucleotidedescribed in the in vitro studies above with the same sequence. ‘3′-THAcounterpart ISIS No.’ refers to the 3′THA conjugated oligonucleotidewith the same sequence and evaluated in the transgenic mice study above.

TABLE 26 ISIS oligonucleotides tested in CD1 mice tolerability studyUncon- jugated 3′-THA ISIS parent counterpart SEQ No. ISIS No. ISIS NoSequence ID NO 863576 848583 863433 GGACATCGGCACATTG 1223 863633 848833863490 AATAATCTCATGTCAG 1016 863655 849040 863512 GTTATTATTGAATGGT  955863670 849171 863527 CTGTAGGAAAGTCTCA 1195 863681 849236 863538CAACATCAAATTCTGC  353

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, creatinine, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, NY). The results are presented in the Tablebelow. Treatment with the ISIS oligonucleotides did not cause anychanges in the levels of any of the liver or kidney function markersoutside the expected range for antisense oligonucleotides.

TABLE 27 Plasma chemistry markers in CD1 mice plasma at Day 45 ALT ASTALB BUN CRE TBIL (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 25 412.6 20.1 0.07 0.13 ISIS 863576 125 134 2.8 24.5 0.10 0.14 ISIS 863633 3560 2.6 21.8 0.10 0.14 ISIS 863655 77 84 2.4 20.0 0.06 0.13 ISIS 86367025 61 2.7 22.5 0.09 0.15 ISIS 863681 27 47 2.8 26.5 0.12 0.14

Hematology Assays

Blood obtained from all mice groups were analyzed for RBC, WBC,platelets, neutrophils, lymphocytes, and monocyte counts, as well ashemoglobin, HCT and MCV levels. The results are presented in the Tablebelow. Treatment with the ISIS oligonucleotides did not cause anychanges in the levels of any of the hematology markers outside theexpected range for antisense oligonucleotides.

TABLE 28 Blood cell count in CD1 mice at Day 45 RBC Platelets WBCNeutro- Lympho- Mono- (×10⁶/ (×10³/ (×10³/ phils cytes cytes μL) μL) μL)(×10³/μL) (×10³/μL) (×10³/μL) PBS 9.0 1288 11.3 12.6 9128 515 863576 8.31251 9.4 18.8 6643 316 863633 9.4 1338 10.1 13.5 8009 698 863655 8.91320 8.0 10.0 5919 473 863670 9.7 1335 8.3 21.3 6514 402 863681 9.5 13297.6 11.0 6376 310

TABLE 29 Hematology markers in CD1 mice at Day 45 Hemoglobin HCT MCV(g/dL) (%) (fL) PBS 14.0 43 48 863576 12.9 40 48 863633 14.6 45 49863655 14.1 44 50 863670 14.7 44 46 863681 14.6 45 47

Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured. Body weights weremeasured every week and are presented in the Table below. Organ weightswere measured and the data is also presented in the Table below. Theresults indicate that effect of treatment with antisenseoligonucleotides on body and organ weights was within the expected rangefor antisense oligonucleotides.

TABLE 30 Body weights (g) in CD1 mice Week Week Week Week Week WeekBaseline 1 2 3 4 5 6 (day 1) (day 8) (day 15) (day 22) (day 29) (day 36)(day 43) PBS 46 48 49 48 50 49 49 863576 29 33 35 36 38 38 39 863633 2732 33 34 36 37 39 863655 28 33 33 35 37 38 39 863670 27 31 32 33 36 3638 863681 28 33 33 35 37 38 40

TABLE 31 Organ weights (g) in CD1 mice Liver Spleen Kidney PBS 2.25 0.160.63 863576 2.32 0.19 0.59 863633 2.25 0.11 0.50 863655 2.55 0.15 0.61863670 1.89 0.11 0.51 863681 2.25 0.10 0.47

Study 2 Treatment

Groups of male CD1 mice were injected subcutaneously once a week for 6weeks with 15 mg/kg of ISIS oligonucleotides. One group of male CD1 micewas injected subcutaneously once a week for 6 weeks with PBS. Mice wereeuthanized 48 hours after the last dose, and organs and plasma wereharvested for further analysis.

The ISIS oligonucleotides tested are presented in the Table below.‘Unconjugated parent ISIS No.’ refers to the ISIS oligonucleotidedescribed in the in vitro studies above with the same sequence. ‘3′-THAcounterpart ISIS No.’ refers to the 3′THA conjugated oligonucleotidewith the same sequence and described in the transgenic mice study above.

TABLE 32 ISIS oligonucleotides tested in CD1 mice tolerability studyUncon- jugated 3′-THA ISIS parent counterpart SEQ No. ISIS No. ISIS NoSequence ID NO 863568 848542 863425 GACAAGTCGGAACCAT 1528 863581 848597863438 ACGGAACAAGAGCTCA 1071 863582 848598 863439 CACGGAACAAGAGCTC 1147863587 848630 863444 CAGAAAGCTAAGCCTC 1149

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, creatinine, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, NY).

The results are presented in the Table below. Treatment with the ISISoligonucleotides did not cause any changes in the levels of any of theliver or kidney function markers outside the expected range forantisense oligonucleotides.

TABLE 33 Plasma chemistry markers in CD1 mice plasma at Day 45 ALT ASTALB BUN CRE TBIL (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 25 512.6 25.8 0.10 0.12 ISIS 863568 72 67 2.6 20.4 0.10 0.14 ISIS 863581 7477 2.7 25.0 0.10 0.14 ISIS 863582 71 89 2.8 24.4 0.11 0.13 ISIS 86358760 60 2.5 24.6 0.11 0.10

Hematology Assays

Blood obtained from all mice groups were analyzed for RBC, WBC,platelets, neutrophils, lymphocytes, and monocyte counts, as well ashemoglobin, HCT and MCV levels. The results are presented in the Tablebelow. Treatment with the ISIS oligonucleotides did not cause anychanges in the levels of any of the hematology markers outside theexpected range for antisense oligonucleotides.

TABLE 34 Blood cell count in CD1 mice at Day 45 RBC Platelets WBCNeutro- Lympho- Mono- (×10⁶/ (×10³/ (×10³/ phils cytes cytes μL) μL) μL)(×10³/μL) (×10³/μL) (×10³/μL) PBS 9.6 853 3.9 12 3174 129 863568 10.3859 7.8 15 6060 347 863581 9.4 812 8.0 17 6162 359 863582 9.1 868 10.521 6671 713 863587 9.6 903 9.8 10 8233 344

TABLE 35 Hematology markers in CD1 mice at Day 45 Hemoglobin HCT MCV(g/dL) (%) (fL) PBS 14.5 44 46 863568 16.2 47 46 863581 14.5 43 46863582 13.5 41 45 863587 15.0 45 47

Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured. Body weights weremeasured every week and are presented in the Table below. Organ weightswere measured and the data is also presented in the Table below. Theresults indicate that effect of treatment with antisenseoligonucleotides on body and organ weights was within the expected rangefor antisense oligonucleotides.

TABLE 37 Organ weights (g) in CD1 mice Liver Spleen Kidney PBS 2.06 0.110.59 863568 2.46 0.10 0.54 863581 2.23 0.13 0.56 863582 2.52 0.16 0.63863587 2.94 0.16 0.64

TABLE 36 Body weights (g) in CD1 mice Week Week Week Week Week WeekBaseline 1 2 3 4 5 6 (day 1) (day 9) (day 16) (day 23) (day 30) (day 37)(day 43) PBS 30 33 36 36 37 39 39 863568 29 32 34 34 36 39 38 863581 2933 35 36 36 39 39 863582 31 34 36 36 37 40 41 863587 30 35 37 37 39 4143

Study 3 Treatment

Groups of male CD1 mice were injected subcutaneously once a week for 6weeks with 5 mg/kg or 15 mg/kg of ISIS oligonucleotides. One group ofmale CD1 mice was injected subcutaneously once a week for 6 weeks withPBS. Mice were euthanized 48 hours after the last dose, and organs andplasma were harvested for further analysis.

The ISIS oligonucleotides tested are presented in the Table below.‘Unconjugated parent ISIS No.’ refers to the ISIS oligonucleotidedescribed in the in vitro studies above with the same sequence. ‘3′-THAcounterpart ISIS No.’ refers to the 3′THA conjugated oligonucleotidewith the same sequence and described in the transgenic mice study above.

TABLE 38 ISIS oligonucleotides tested in CD1 mice tolerability studyUncon- jugated 3′-THA ISIS parent counterpart SEQ No. ISIS No. ISIS NoSequence ID NO 845219 466847 863413 GAGGTATCCCCGGCGG 1096 863577 848584863434 ACGGACATCGGCACAT  377 863579 848593 863436 AGCTCAATAAAAGTCA  763863637 848849 863494 CAAATTATAGCAGCCA 1017 863682 849239 863539GAAGACGGAGTAAGGC 1352

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, creatinine, and BUNwere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, NY).

The results are presented in the Table below. ISIS oligonucleotides thatcaused changes in the levels of any of the liver or kidney functionmarkers outside the expected range for antisense oligonucleotides wereexcluded in further studies.

TABLE 39 Plasma chemistry markers in CD1 mice plasma at Day 45 CRE TBILDose ALT AST ALB BUN (mg/ (mg/ (mg/kg) (IU/L) (IU/L) (g/dL) (mg/dL) dL)dL) PBS — 26 62 2.5 19.5 0.08 0.18 845219 5 96 64 2.4 19.3 0.08 0.15 15187 115 2.3 22.0 0.07 0.17 863577 5 176 160 2.5 17.2 0.09 0.13 15 621492 2.5 15.6 0.10 0.20 863579 5 50 50 2.5 19.9 0.10 0.13 15 87 70 2.420.3 0.11 0.13 863637 5 321 301 1.7 19.3 0.05 0.19 15 508 472 2.4 20.50.09 0.22 863682 5 306 156 2.3 20.0 0.06 0.13 15 373 212 2.4 21.0 0.060.14

Hematology Assays

Blood obtained from all mice groups were analyzed for RBC, WBC,platelets, neutrophils, lymphocytes, and monocyte counts, as well ashemoglobin, HCT and MCV levels. The results are presented in the Tablebelow. ISIS oligonucleotides that caused changes in the levels of any ofthe hematology markers outside the expected range for antisenseoligonucleotides were excluded in further studies.

TABLE 40 Blood cell count in CD1 mice at Day 45 Dose RBC Platelets WBCNeutrophils Lymphocytes Monocytes (mg/kg) (×10⁶/μL) (×10³/μL) (×10³/μL)(×10³/μL) (×10³/μL) (×10³/μL) PBS — 9.4 1218 8.8 13 7078 367 845219 59.4 1349 7.2 13 5749 444 15 9.3 1263 7.2 13 5664 511 863577 5 9.2 14018.0 17 5851 650 15 8.2 1299 10.0 10 7718 1034 863579 5 10.0 1252 9.0 166876 587 15 9.6 1256 8.2 11 6645 415 863637 5 9.0 956 5.7 14 4266 448 159.3 843 6.8 13 5044 696 863682 5 9.4 1040 5.9 13 4581 447 15 9.0 800 8.38 6757 694

TABLE 41 Hematology markers in CD1 mice at Day 45 Dose Hemoglobin HCTMCV (mg/kg) (g/dL) (%) (fL) PBS — 14.1 45 48 845219 5 14.2 45 49 15 13.843 46 863577 5 13.9 45 49 15 12.2 39 48 863579 5 14.9 47 48 15 15.3 4850 863637 5 13.5 42 47 15 13.8 43 47 863682 5 14.5 45 48 15 13.6 43 48

Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured. Body weights weremeasured every week and are presented in the Table below. Organ weightswere measured and the data is also presented in the Table below. Theresults indicate that effect of treatment with the antisenseoligonucleotides on body and organ weights was within the expected rangefor antisense oligonucleotides.

TABLE 42 Body weights (g) in CD1 mice Dose Baseline Week 1 Week 2 Week 3Week 4 Week 5 Week 6 (mg/kg) (day 1) (day 8) (day 15) (day 22) (day 29)(day 36) (day 42) PBS — 27 30 32 32 33 35 35 845219 5 28 33 34 35 37 4039 15 29 33 34 35 36 38 38 863577 5 26 31 32 33 34 37 35 15 27 31 31 3234 36 35 863579 5 28 32 33 32 36 38 36 15 27 32 33 32 36 38 37 863637 528 33 34 35 36 37 38 15 27 32 34 34 36 38 38 863682 5 26 32 33 32 35 3736 15 27 31 32 33 34 36 36

TABLE 43 Organ weights (g) in CD1 mice Dose (mg/kg) Liver Spleen KidneyPBS — 1.84 0.08 0.50 845219 5 2.42 0.13 0.54 15 2.41 0.15 0.54 863577 52.47 0.11 0.58 15 2.62 0.15 0.59 863579 5 2.53 0.13 0.57 15 2.82 0.120.54 863637 5 2.38 0.17 0.52 15 2.67 0.22 0.58 863682 5 1.79 0.11 0.5415 2.13 0.21 0.53

Example 6: Tolerability of ISIS Oligonucleotides Targeting Human PCSK9in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. The rats were treated with ISIS antisenseoligonucleotides from the studies described in the Examples above andevaluated for changes in the levels of various plasma chemistry markers.

Treatment

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycleand fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4Sprague-Dawley rats each were injected subcutaneously once a week for 6weeks with 15 mg/kg of ISIS oligonucleotide. Forty eight hours after thelast dose, rats were euthanized and organs and plasma were harvested forfurther analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Plasma levelsof ALT (alanine transaminase) and AST (aspartate transaminase) weremeasured and the results are presented in the Table below expressed inIU/L. Plasma levels of bilirubin (TBIL), BUN, albumin (ALB), andcreatinine (CRE) were also measured using the same clinical chemistryanalyzer and the results are also presented in the Table below expressedin mg/dL. Plasma levels of albumin were also measured using the sameclinical chemistry analyzer and the results are also presented in theTable below expressed in g/dL.

TABLE 44 Liver function markers in Sprague-Dawley rats ALT AST ALB BUNCRE TBIL (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 20 64 3.1 130.2 0.1 863568 40 70 3.5 14 0.2 0.2 863576 39 94 3.7 16 0.3 0.1 86357933 91 3.3 14 0.2 0.1 863581 36 154 2.8 18 0.3 0.2 863582 38 122 2.9 200.3 0.1 863587 38 87 3.0 12 0.2 0.1 863633 26 78 3.0 16 1 0.3 0.1 86365527 84 3.1 15 0.2 0.1 863670 30 91 3.2 15 0.2 0.1 863681 29 81 3.2 14 0.20.1

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,urine levels of blood creatinine and total protein were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, NY). Results are presented in the Table below, expressed inmg/dL. ISIS oligonucleotides that caused changes in the levels of any ofthe kidney function markers outside the expected range for antisenseoligonucleotides were excluded in further studies. ‘N/A’ indicates thatdata is not available for that group.

TABLE 45 Kidney function markers (mg/dL) in Sprague-Dawley rats CRE TP(mg/dL) (mg/dL) PBS 79 59 863568 111 137 863576 N/A N/A 863579 93 117863581 54 75 863582 71 91 863587 64 86 863633 100 85 863655 85 117863670 89 104 863681 144 142

Body and Organ Weights

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured. Body weights weremeasured every week and are presented in the Table below. Organ weightswere measured and the data is also presented in the Table below. Theresults indicate that effect of treatment with antisenseoligonucleotides on body and organ weights was within the expected rangefor antisense oligonucleotides.

TABLE 46 Body weight (g) Week 4 Week 5 Week 6 Baseline Week 1 Week 2Week 3 (day (day (day (day 1) (day 8) (day 15) (day 22) 29) 36) 43) PBS201 267 344 389 433 451 470 863568 200 265 334 371 404 419 435 863576272 327 361 389 417 424 435 863579 264 313 357 379 407 420 430 863581195 258 329 361 397 411 430 863582 204 273 350 387 423 440 458 863587205 273 353 398 439 459 474 863633 271 319 361 377 401 406 417 863655195 256 330 366 406 417 435 863670 189 249 319 351 384 394 406 863681275 312 364 386 415 426 435

TABLE 47 Organ weights (g) Liver Spleen Kidney PBS 12 0.8 3.4 863568 161.2 3.3 863576 12 1.3 3.0 863579 11 1.0 3.0 863581 13 2.0 3.1 863582 142.1 3.5 863587 14 1.1 3.4 863633 12 1.6 3.2 863655 11 1.7 3.4 863670 121.0 2.9 863681 12 1.1 3.3

Example 7: Effect of Antisense Inhibition of PCSK9 in Transgenic MouseModel

A transgenic mouse model developed at UCI and has never been describedin the literature. The human PCSK9 genomic construct contained in fosmidABC7-611722G24 was restricted with Nhel to produce a DNA fragmentcontaining the entire genomic sequence as well as 8 Kb of 5′ and 0.4 Kbof 3′ noncoding sequence. PCSK9 transgenic mice were produced by randominsertion via micronucleus injection. Progeny expressed human PCSK9 mRNAin the liver and secreted human PCSK9 plasma protein.

Treatment

Transgenic mice were maintained on a 12-hour light/dark cycle and werefed ad libitum normal Purina mouse chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. Antisense oligonucleotides (ASOs) were prepared in bufferedsaline (PBS) and sterilized by filtering through a 0.2 micron filter.Oligonucleotides were dissolved in 0.9% PBS for injection.

The Tg mice were divided into 27 groups of 3 mice each. Groups receivedsubcutaneous injections of ISIS oligonucleotide at a dose of 0.25 mg/kg,1.00 mg/kg, or 5.00 mg/kg once per week for 4 weeks. One group of 4 micereceived subcutaneous injections of PBS once per week for 4 weeks. Thesaline-injected group served as the control group to whicholigonucleotide-treated groups were compared.

RNA Analysis

On day 26, RNA was extracted from liver for real-time PCR analysis ofmeasurement of mRNA expression of PCSK9. Results are presented aspercent change of mRNA, relative to PBS control, normalized withRIBOGREEN®. As shown in the Table below, treatment with ISIS antisenseoligonucleotides resulted in significant reduction of PCSK9 mRNA incomparison to the PBS control.

TABLE 48 Percent inhibition of PCSK9 mRNA in the transgenic mice liverrelative to the PBS control Weekly Dose % (mg/kg) Inhibition ED₅₀ 8635680.25 26 0.5 1 76 5 95 863579 0.25 9 1.4 1 41 5 84 863581 0.25 12 0.6 173 5 80 863582 0.25 32 0.5 1 71 5 91 863587 0.25 27 0.5 1 71 5 87 8636330.25 60 0.2 1 96 5 99 863655 0.25 37 0.3 1 94 5 99 863670 0.25 0 1.0 162 5 87 863681 0.25 25 0.9 1 45 5 94

Protein Analysis

PCSK9 plasma protein was measured by a human-specific ELISA kit (R&DSystems). Results are presented as percent change of protein levels,relative to PBS control. As shown in the Table below, treatment withISIS antisense oligonucleotides resulted in significant reduction ofPCSK9 plasma protein levels in comparison to the PBS control.

TABLE 49 Percent inhibition of PCSK9 plasma protein in the transgenicmice relative to the PBS control Weekly Dose % (mg/kg) Inhibition ED₅₀863568 0.25 37 0.4 1 74 5 97 863579 0.25 5 1.5 1 41 5 81 863581 0.25 310.6 1 59 5 91 863582 0.25 25 0.5 1 73 5 95 863587 0.25 33 0.4 1 77 5 93863633 0.25 50 0.3 1 97 5 99 863655 0.25 44 0.3 1 93 5 99 863670 0.25 231.6 1 38 5 72 863681 0.25 28 0.8 1 54 5 89

LDL-Cholesterol Levels

Levels of LDL-cholesterol in the plasma were measured by an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY).Levels of LDL receptor (LDLr) protein in the liver were also measured.Results are presented as percent change of levels, relative to PBScontrol. As shown in the Table below, treatment with several ISISantisense oligonucleotides resulted in significant reduction of levelsof LDL-cholesterol in the plasma in comparison to the PBS control.Correspondingly, it was observed that treatment with ISIS antisenseoligonucleotides resulted in dose-dependent liver LDLr protein increasesin the mice.

TABLE 50 Inhibition of LDL-cholesterol levels in the plasma relative tothe PBS control Weekly Dose % (mg/kg) inhibition 863568 0.25 7 1 41 5 59863579 0.25 0 1 15 5 33 863581 0.25 30 1 17 5 41 863582 0.25 23 1 28 550 863587 0.25 20 1 38 5 38 863633 0.25 27 1 66 5 71 863655 0.25 29 1 585 74 863670 0.25 19 1 0 5 39 863681 0.25 21 1 23 5 35

TABLE 51 Percent increase of liver LDLr in the transgenic mice relativeto the PBS control Weekly Dose % (mg/kg) increase 863568 0.25 3 1 61 5245 863579 0.25 −46 1 −45 5 132 863581 0.25 −44 1 6 5 73 863582 0.25 −191 93 5 191 863587 0.25 −6 1 −10 5 95 863633 0.25 −22 1 131 5 269 8636550.25 0 1 129 5 207 863670 0.25 9 1 81 5 78 863681 0.25 123 1 360 5 222

Example 8: Measurement of Viscosity of Antisense OligonucleotidesTargeting Human PCSK9

The viscosity of select antisense oligonucleotides from the studiesdescribed above was measured with the aim of screening out antisenseoligonucleotides which have a viscosity of more than 40 centipoise (cP).Oligonucleotides having a viscosity greater than 40 cP would have lessthan optimal viscosity.

Oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL ofwater was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part (75 μL) of the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometer was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in the Table below, where theconcentration of each antisense oligonucleotide was 350 mg/ml, andindicate that all the antisense oligonucleotides solutions were optimalin their viscosity under the criterion stated above.

TABLE 52 Viscosity of antisense oligonucleotides targeting human PCSK9Viscosity Concentration ISIS No (cp) (mg/mL) 863568 39 325 863576 20 325863579 19 325 863581 25 300 863582 17 300 863587 14 325 863633 12 325863655 10 325 863670 21 325 863681 15 325

Example 9: Effect of ISIS Antisense Oligonucleotides Targeting HumanPCSK9 in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesselected from studies described in the Examples above. Antisenseoligonucleotide efficacy and tolerability, as well as theirpharmacokinetic profile in the liver and kidney, were evaluated.

At the time this study was undertaken, the cynomolgus monkey genomicsequence was not available in the National Center for BiotechnologyInformation (NCBI) database; therefore, cross-reactivity with thecynomolgus monkey gene sequence could not be confirmed. Instead, thesequences of the ISIS antisense oligonucleotides used in the cynomolgusmonkeys was compared to a rhesus monkey sequence for homology. It isexpected that ISIS oligonucleotides with homology to the rhesus monkeysequence are fully cross-reactive with the cynomolgus monkey sequence aswell. The human antisense oligonucleotides tested are cross-reactivewith the rhesus genomic sequence (the complement of GENBANK AccessionNo. NW_005092960.1 truncated from nucleotides 83474000 to 83501000,designated herein as SEQ ID NO: 1544). The greater the complementaritybetween the human oligonucleotide and the rhesus monkey sequence, themore likely the human oligonucleotide can cross-react with the rhesusmonkey sequence. The start and stop sites of each oligonucleotide to SEQID NO: 1544 is presented in the Table below. “Start site” indicates the5′-most nucleotide to which the gapmer is targeted in the rhesus monkeygene sequence.

TABLE 53 Target Target SEQ ISIS Start Stop ID No Site Mismatches Site NO863568 25252 1 25267 1528 863579 25510 2 25525 763 863581 25520 1 255351071 863582 25521 1 25536 1147 863587 25712 2 25727 1149 863633 3911 03926 1016 863655 10464 1 10479 955 863670 15306 0 15321 1195 86368117270 0 17285 353

Treatment

Prior to the study, the monkeys were kept in quarantine during which theanimals were observed daily for general health. The monkeys were 2-4years old and weighed 2-4 kg. Nine groups of 5 randomly assigned malecynomolgus monkeys each were injected subcutaneously with ISISoligonucleotide or PBS in clockwise rotation between four differentsites on the backs of the monkeys (i.e. left, right, top, and bottom) oneach day of dosing; one site per dose. The monkeys were dosed twice aweek (days 1, 5, 9, and 14) for the first two weeks, and thensubsequently once a week for 10 weeks (days 21, 28, 35, 42, 49, 56, 63,70, 77, and 84) with 10 mg/kg of ISIS oligonucleotide. A control groupof 5 cynomolgus monkeys was injected with PBS in a similar manner andserved as the control group.

During the study period, the monkeys were observed twice daily for signsof illness or distress. Any animal experiencing more than momentary orslight pain or distress due to the treatment, injury or illness wastreated by the veterinary staff with approved analgesics or agents torelieve the pain after consultation with the Study Director. Any animalin poor health or in a possible moribund condition was identified forfurther monitoring and possible euthanasia. Scheduled euthanasia of theanimals was conducted on day 86 approximately 48 hours post-dose byexsanguination while under deep anesthesia. The protocols described inthe Example were approved by the Institutional Animal Care and UseCommittee (IACUC).

Target Reduction RNA Analysis

On day 86, RNA was extracted from liver for real-time PCR analysis ofmeasurement of mRNA expression of PCSK9. Results are presented aspercent change of mRNA, relative to PBS control, normalized withRIBOGREEN®. As shown in the Table below, treatment with ISIS antisenseoligonucleotides, ISIS 863633, ISIS 863670, and ISIS 863681, which hadno mismatches with the rhesus gene, resulted in significant reduction ofPCSK9 mRNA in comparison to the PBS control.

TABLE 54 Percent inhibition of PCSK9 mRNA in the cynomolgus monkey liverrelative to the PBS control % inhibition 863655 46 863633 88 863568 74863579 35 863582 55 863581 53 863587 36 863670 84 863681 87

Protein Analysis

Approximately 1 mL of blood was collected from all available animals onday −7 (pre-treatment), Days 16, 30, 58, and 86 (approximately 48 hourspost-dose on Days 14, 28, 56, and 84, respectively) and placed in tubescontaining the potassium salt of EDTA. The tubes were centrifuged (3000rpm for 10 min at 4° C.) to obtain plasma. The results are presented inthe Table below as a percentage increase or decrease over the levels atDay −7 (pre-treatment levels). As shown in the Table below, treatmentwith ISIS antisense oligonucleotides, ISIS 863633, ISIS 863670, and ISIS863681, which had no mismatches with the rhesus gene, resulted insignificant reduction of PCSK9 protein levels.

Liver tissue was also analyzed for levels of LDL receptor (LDLr) proteininduction levels. The results are presented in the Table below. The datademonstrates that treatment with the three antisense oligonucleotidesthat are homologous to the rhesus monkey genomic sequence resulted inhepatic LDLr protein induction after 12 weeks of treatment.

TABLE 55 Plasma protein levels (% of pre-treatment levels) in thecynomolgus monkey Day 16 Day 30 Day 86 PBS 34 36 18 863655 −13 −2 −36863633 −90 −91 −91 863568 −19 −14 −22 863579 34 36 18 863582 8 2 35863581 16 8 11 863587 −3 1 −12 863670 −79 −83 −86 863681 −80 −82 −86

TABLE 56 Hepatic LDLr protein levels (% of PBS control) in thecynomolgus monkey % increase 863655 98 863633 399 863568 243 863579 163863582 115 863581 156 863587 158 863670 521 863681 417

Plasma Lipid Levels

To evaluate the effect of ISIS oligonucleotides on total cholesterol,LDL-Cholesterol, HDL-cholesterol, and triglyceride levels, monkeys werefasted overnight prior to blood collection. Approximately 1.7 mL ofblood samples were collected from all the study groups. Blood wascollected in tubes without anticoagulant for serum separation. The tubeswere kept at room temperature for a minimum of 90 minutes and thencentrifuged at 3,000 rpm for 10 minutes at room temperature to obtainserum. Levels were measured using a Toshiba 200FR NEO chemistry analyzer(Toshiba Co., Japan). The data demonstrates that treatment with thethree antisense oligonucleotides with homology with the rhesus monkeygenomic sequence resulted in significant reduction of plasma totalcholesterol and LDL-cholesterol. As expected, reduction of human PCSK9levels did not affect plasma HDL-cholesterol or triglyceride levels.

TABLE 57 Total LDL- HDL- cholesterol cholesterol cholesterolTriglycerides PBS 146 37 110 71 863655 152 73 71 46 863633 115 17 99 57863568 128 32 99 51 863579 109 44 71 45 863582 137 38 103 37 863581 17451 122 34 863587 140 45 102 35 863670 95 17 83 45 863681 109 19 99 29

Tolerability Studies Clinical Chemistry Parameters

To evaluate the effect of ISIS oligonucleotides on hepatic function,monkeys were fasted overnight prior to blood collection. Approximately1.7 mL of blood samples were collected from all the study groups. Bloodwas collected in tubes without anticoagulant for serum separation. Thetubes were kept at room temperature for a minimum of 90 minutes and thencentrifuged at 3,000 rpm for 10 minutes at room temperature to obtainserum. Levels of total bilirubin (TBIL), aspartate aminotransferase(AST), alanine aminotransferase (ALT), and albumin (ALB) were measuredusing a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Theresults indicate that antisense oligonucleotides had no effect on liverfunction outside the expected range for antisense oligonucleotides.Specifically, ISIS 863633 was observed to be well tolerated.

TABLE 58 Plasma Chemistry markers in cynomolgus monkey on Day 86 ALT ASTBilirubin ALB (IU/L) (IU/L) (mg/dL) (g/dL) PBS 65 64 0.2 4.1 863655 6359 0.3 3.5 863633 47 70 0.3 4.0 863568 44 64 0.3 4.1 863579 50 52 0.24.1 863582 42 57 0.2 4.2 863581 55 61 0.3 4.2 863587 110 76 0.3 3.9863670 42 64 0.3 4.1 863681 62 60 0.3 4.1

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,monkeys were fasted overnight prior to blood collection. Approximately1.7 mL of blood samples were collected from all the study groups. Bloodwas collected in tubes without anticoagulant for serum separation. Thetubes were kept at room temperature for a minimum of 90 minutes and thencentrifuged at 3,000 rpm for 10 minutes at room temperature to obtainserum. Levels of BUN and creatinine were measured using a Toshiba 200FRNEO chemistry analyzer (Toshiba Co., Japan). Results are presented inthe Table below, expressed in mg/dL.

The plasma chemistry data indicate that most of the ISISoligonucleotides did not have any effect on the kidney function outsidethe expected range for antisense oligonucleotides. Specifically, ISIS863633 was observed to be well tolerated.

TABLE 59 Plasma BUN and creatinine levels (mg/dL) in cynomolgus monkeysat day 86 BUN Creatinine PBS 31 0.8 863655 26 0.8 863633 27 0.8 86356827 0.9 863579 24 0.8 863582 24 0.8 863581 27 0.8 863587 24 0.8 863670 261.0 863681 23 0.8

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys onhematologic parameters, approximately 0.5 mL of blood was collected fromeach of the available study animals in tubes containing K₂-EDTA. Sampleswere analyzed for red blood cell (RBC) count, white blood cells (WBC)count, individual white blood cell counts, such as that of monocytes,neutrophils, lymphocytes, as well as for platelet count, hemoglobincontent and hematocrit, using an ADVIA120 hematology analyzer (Bayer,USA). The data is presented in the Tables below.

The data indicate the oligonucleotides did not cause any changes inhematologic parameters outside the expected range for antisenseoligonucleotides at this dose. Specifically, ISIS 863633 was observed tobe well tolerated.

TABLE 60 Blood cell counts in cynomolgus monkeys on day 86 RBC PlateletsWBC Neutro- Lympho- Mono- (×10⁶/ (×10³/ (×10³/ phils cytes cytes μL) μL)μL) (×10³/μL) (×10³/μL) (×10³/μL) PBS 5.5 340 9.0 3.1 5.4 0.3 863655 5.6361 9.7 3.7 5.4 0.3 863633 5.6 395 11.4 4.5 6.5 0.3 863568 5.8 409 12.1602 5.4 0.3 863579 5.9 390 8.0 2.2 5.3 0.3 863582 5.6 333 8.9 3.0 5.50.3 863581 5.8 382 8.2 2.4 5.3 0.3 863587 6.0 348 8.8 2.3 6.0 0.2 8636705.4 417 10.7 4.3 5.9 0.3 863681 5.6 408 9.0 3.1 5.5 0.2

TABLE 61 Hemoglobin HCT (g/dL) (%) PBS 12.8 45 863655 13.0 43 86363312.9 43 863568 13.0 44 863579 13.5 46 863582 12.4 43 863581 13.0 44863587 13.5 46 863670 12.7 43 863681 12.7 43

Overall, the results of the study indicate that ISIS 863633 is the mostpotent and well tolerated compound of those tested for inhibiting PCSK9and is an important candidate for the treatment of cardiovasculardiseases, specifically for lowering LDL-cholesterol levels.

Example 10: Comparative Inhibition of Antisense OligonucleotidesTargeting Human PCSK9 in a Dose Response Assay

The unconjugated parent oligonucleotides of the antisenseoligonucleotides tested in the monkey study were compared withpreviously designed compounds. ISIS 848542, ISIS 848593, ISIS 848597,ISIS 848598, ISIS 848630, ISIS 848833, ISIS 849040, ISIS 849171, andISIS 849236 were tested for efficacy with ISIS 405879 and ISIS 405995,which have previously been determined to be some of the most potentantisense compounds in vitro (see e.g., U.S. Pat. No. 8,084,437), aswell as ISIS 431131 and ISIS 480604, which have been previouslydescribed in U.S. Pat. No. 9,127,276.

Cells were plated at a density of 10,000 cells per well and 39.1 nM,156.25 nM, 625 nM, 2,500 nM, and 10,000 nM concentrations of antisenseoligonucleotide was added to the medium for free uptake ofoligonucleotide by the cells. After a treatment period of approximately48 hours, RNA was isolated from the cells and PCSK9 mRNA levels weremeasured by quantitative real-time PCR. Human PCSK9 primer probe setfrom ABI (ID #Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of PCSK9,relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. PCSK9 mRNA levels were reduced in a dose-dependentmanner in antisense oligonucleotide treated cells. ISIS 848542, ISIS848593, ISIS 848597, ISIS 848598, ISIS 848630, ISIS 848833, ISIS 849040,ISIS 849171, and ISIS 849236 demonstrated superior efficacy compared toall previously disclosed oligonucleotides.

TABLE 62 39.1 156.25 625.0 2500.0 10000.0 IC₅₀ ISIS No nM nM nM nM nM(μM) 405879 0 0 0 0 0 >10 405995 0 0 0 0 0 >10 431131 0 3 0 0 5 >10480604 0 1 19 31 36 >10 848542 9 37 70 83 88 0.4 848593 19 36 51 53 691.1 848597 41 67 78 86 91 0.04 848598 41 64 76 85 90 0.1 848630 0 17 5979 85 0.9 848833 4 66 88 93 94 0.2 849040 41 93 98 99 98 <0.03 849171 052 86 94 96 0.3 849236 0 26 71 89 95 0.6

1-66. (canceled)
 67. A compound or a pharmaceutically acceptable salt thereof, comprising a modified oligonucleotide 12 to 30 linked nucleosides in length having a nucleobase sequence comprising at least 12 contiguous nucleobases of the nucleobase sequence of SEQ ID NO:
 1016. 68. The compound of claim 67, wherein at least one of the nucleosides of the modified oligonucleotide comprises a cEt sugar.
 69. The compound of claim 67, wherein the modified oligonucleotide comprises a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of three linked nucleosides; and a 3′ wing segment consisting of three linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment and the 3′ wing segment comprise cEt sugars; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.
 70. The compound of claim 67, further comprising a conjugate group and a conjugate linker.
 71. The compound of claim 71, wherein the conjugate group is attached to the modified oligonucleotide at the 5′-end of the modified oligonucleotide.
 72. The compound of claim 71, wherein the conjugate group is attached to the modified oligonucleotide at the 3′-end of the modified oligonucleotide.
 73. The compound of claim 72, wherein the conjugate group is 5′-Trishexylamino-(THA)-C6GalNAC3.
 74. The compound of claim 67, wherein the pharmaceutically acceptable salt is a sodium salt.
 75. The compound of claim 67, wherein the pharmaceutically acceptable salt is a potassium salt.
 76. A pharmaceutical composition comprising the compound of claim 67, and a pharmaceutically acceptable diluent or carrier.
 77. A pharmaceutical composition comprising the sodium salt of claim 74, and a pharmaceutically acceptable diluent or carrier.
 78. A pharmaceutical composition comprising the potassium salt of claim 75, and a pharmaceutically acceptable diluent or carrier.
 79. A method of treating, preventing, or ameliorating a disease associated with PCSK9 in an individual comprising administering to the individual a compound of claim
 67. 80. The method of claim 79, wherein the disease is selected from hypercholesterolemia, dyslipidemia or mixed dyslipidemia.
 81. The method of claim 79, wherein administering the compound inhibits or reduces LDL-cholesterol levels and total cholesterol levels and improves the induction of hepatic LDL receptor levels.
 82. A method of inhibiting expression of PCSK9 in a cell comprising contacting the cell with a compound of claim
 67. 83. The method of claim 82, wherein the cell is in the liver of an individual.
 84. The method of claim 83, wherein the individual has, or is at risk of having, hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.
 85. A method of reducing or inhibiting LDL-cholesterol levels and total cholesterol levels in an individual having, or at risk of having, a disease associated with PCSK9 comprising administering a compound of claim
 67. 86. The method of claim 85, wherein the individual has, or is at risk of having, hypercholesterolemia, dyslipidemia, or mixed dyslipidemia. 